MVA-gH/gL-PC VACCINE DERIVED ANTIBODIES NEUTRALIZING HUMAN CYTOMEGALOVIRUS INFECTIVTIY AND METHODS THEREOF

ABSTRACT

Disclosed are vaccine-derived neutralizing antibodies (NAbs) for CMV infections and small peptides which define precise recognition elements of the antigens by the NAbs. In certain embodiments, vaccine-derived NAbs may be produced by immunizing a subject with a gH/gL/UL128/UL130/UL131A pentameric glycoprotein complex (gH/gL-PC). In certain embodiments, vaccine-derived NAbs may have properties similar or identical to those of NAbs induced in a subject naturally infected with CMV. Native and non-native small peptides from UL128 and gH have been defined by mapping epitopes and deriving artificial sequences which are minimal recognition elements of vaccine-derived NAbs disclosed herein. These small peptides can be used to elicit vaccine-derived NAbs that prevent CMV entry into susceptible cell types and protect humans from infection and disease. Multivalent vaccines comprising these small peptides and/or epitopes are also disclosed. Kits and methods of using the vaccine-derived NAbs and small peptides disclosed herein including methods of treating or preventing CMV infection in a subject are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of International ApplicationNo. PCT/US2016/051167, filed Sep. 9, 2016, which claims priority to U.S.Provisional Application No. 62/216,939, filed Sep. 10, 2015, which isincorporated herein by reference in its entirety, including drawings.

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with government support under Grant No.R01AI103960-02 awarded by the National Institutes of Health (NIH). TheGovernment has certain rights in the invention.

BACKGROUND

Human cytomegalovirus (HCMV) is the most common infectious cause ofpermanent births defects worldwide, often resulting in auditory andcognitive abnormalities and in rare cases even in multi-organ failureand death (1-4). Congenital HCMV infection occurs in 0.05 to 1% of allpregnancies, and 10 to 25% of congenitally infected newborns developlong-term developmental disabilities (2-6). Annual incidence of HCMVseropositive (HCMV+) infants at birth range from 35,000 in Brazil to40,000 in the United States, and 250,000 in India (5). In fact,persistent newborn medical conditions are more frequently associatedwith congenital HCMV infection than with other well-known childhooddiseases such as trisomy 21, spina bifida, or fetal-alcohol syndrome (2,4, 7-10). Besides its leading role in permanent birth defects, HCMV isalso a major cause of morbidity and mortality in hematopoetic stem celland solid organ transplant recipients (11-13). Based on the societal andfinancial health burden and in the absence of effective treatmentoptions, HCMV has been assigned as one of the highest priority vaccinetargets (14, 15). However, incompletely defined correlates ofprotection, lack of animal models susceptible to HCMV infection,insufficiently powered vaccine trials, and general unawareness, are anumber of obstacles that have hampered the development of an effectiveand safe HCMV vaccine (16).

High titer and durable neutralizing antibodies (NAbs) that blockglycoprotein complex-mediated entry into host cells are thought to beessential to prevent or control congenital HCMV infection. For manydecades, HCMV subunit vaccine research has primarily focused onstimulation of NAbs targeting the major essential envelope glycoproteinB (gB), culminating in the encouraging findings obtained withrecombinant gB admixed in adjuvant MF59 (17). In phase II clinicaltrials, gB/MF59 has been shown to reduce viremia and the need forantiviral therapy in solid organ transplant recipients and providemoderate efficacy of 38-50% to prevent primary infection in young womenof childbearing age (17-20). These findings have spurred interest toimprove vaccine-mediated induction of NAb responses as an approach toimprove protective efficacy beyond that observed with gB/MF59.

In recent years it has been recognized that HCMV entry into fibroblasts(FB) and epithelial/endothelial cells (EpC/EnC) occurs by alternateroutes of entry that are blocked by NAbs of varying potency andcell-type specificity (21-23). HCMV infection of FB depends on the majoressential envelope glycoprotein complexes (gC) gM/gN, gB, and gH/gL (22,23). In contrast to FB entry, HCMV infection of EpC/EnC requires anadditional complex formed by gH/gL, UL128, UL130, and UL131A (PC) (21,24-26). A third gH/gL complex composed of gH/gL/gO appears necessary forentry into both FB and EpC/EnC, though this remains controversial(27-31). NAbs targeting the major gC block both HCMV entry routes (32);however, NAbs recognizing predominantly conformational epitopes formedby two or more of the UL128/130/131A subunits of the PC are unable toprevent FB entry, though they have potency to interfere with EpC/EnCinfection that dramatically exceed that of NAb targeting the major gC(32, 33).

Many vaccine strategies based on either live-attenuated viruses, viralvector systems or purified proteins confirm that the PC is the majortarget of NAbs blocking HCMV infection of EpC/EnC (33-36). All of thesevaccine approaches consistently demonstrate in animal models that the PChas superior immunogenicity to elicit NAbs against EpC/EnC entrycompared to PC subunit subsets (gH/gL or UL128/UL130/UL131A) or gB(33-36). These studies also show that vaccine approaches employing thePC are equally or even more effective than gB-based vaccine strategiesto induce NAbs blocking FB entry (33, 34, 36). Consequently, PC subunitvaccines elicit high titer EpC/EnC specific NAb responses and lesspotent NAbs against FB entry, which is consistent with the NAb responseinduced by HCMV during natural infection (37-39).

Although the mechanisms through which HCMV crosses the placenta arestill debated, cytotrophoblasts (CTB) including their syncytial formsand progenitors are thought to be the key mediators involved in allpotential HCMV vertical transmission routes (40-44). These cells build abridge at the fetal-maternal interface and can be efficiently infectedby HCMV in vitro and in vivo (10, 43-46). In addition, infection of CTBin early gestation often results in placental developmentalabnormalities (44, 46-48). However, NAbs that interfere with HCMVinfection of placental cells are only poorly characterized. A recentstudy has shown that HCMV infection of CTB progenitor cells can beefficiently blocked by NAbs to gB, although NAbs targeting the PC areunable to interfere with CTB progenitor infection (49, 50). WhetherPC-specific NAbs are able to prevent infection of differentiating CTB isunknown.

Accordingly, there remains a need to develop highly effective antibodiesto neutralize CMV infections, particularly HCMV infections.

SUMMARY

In one aspect, the disclosure provided herein relates to avaccine-derived neutralizing antibody (NAb) against cytomegalovirus(CMV). In some embodiments, the vaccine-derived NAb is against human CMV(HCMV). The vaccine-derived NAb comprises a variable heavy regioncomprising a CDR1_(VH) sequence, a CDR2_(VH) sequence, and a CDR3_(VH)sequence; a variable light region comprising a CDR1_(VL) sequence, aCDR2_(VL) sequence, and a CDR3_(VL) sequence; wherein thevaccine-derived NAb is produced in response to a recombinant CMVpentameric complex comprising gH, gL, UL128, UL130, and UL131A(“gH/gL-PC”).

In some embodiments, the vaccine-derived NAb is similar or identical toa NAb induced in a subject naturally infected with CMV in one or moreproperties selected from the group consisting of cell-type specificity,neutralization potency, minimal antigen recognition, and frequency torecognize antigenic sites. In some embodiments, the vaccine-derived NAbprevents cell-to-cell spread of CMV, syncytia formation in epithelialcells, or both. In some embodiments, the vaccine-derived NAb has apositive correlation between neutralizing potency and binding affinityof one or more cell surface subunits of the pentameric complex.

In some embodiments, the vaccine-derived NAb specifically binds one ormore linear epitopes on the recombinant CMV pentameric complex. Thelinear epitope is on UL128 of the recombinant CMV pentameric complex,and the linear epitope on UL128 may comprise an amino acid sequencerepresented by SEQ ID NO: 177 (KRLDVCRAKMGYM). Alternatively, the linearepitope is on gH of the recombinant CMV pentameric complex, and thevaccine-derived NAb neutralizes CMV infection of epithelial cells butnot CMV infection of fibroblasts.

In some embodiments, the vaccine-derived NAb specifically binds one ormore conformational epitopes on the recombinant CMV pentameric complex.The vaccine-derived NAb specifically binds to one or more conformationalepitopes composed of UL128/UL130/UL131A or UL130/UL131A subunits of therecombinant CMV pentameric complex, and neutralizes CMV infection ofepithelial cells, endothelial cells, primary placental cytotrophoblastcells or a combination thereof.

In some embodiments, the vaccine-derived NAb specifically binds to oneor more conformational epitopes on gH or gH/gL, and the vaccine-derivedNAb prevents CMV infection of fibroblasts, epithelial cells, endothelialcells, cytotrophoblasts or a combination thereof.

In some embodiments, the vaccine-derived NAb has a CDR1_(VH) sequenceselected from the group consisting of SEQ ID NOs. 3, 11, 19, 27, 35, 43,51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171,and sequences sharing at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity to SEQ ID NOs. 3, 11, 19, 27, 35, 43, 51,59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147, 155, 163, 171, aCDR2_(VH) sequence selected from the group consisting of SEQ ID NOs. 4,12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132,140, 148, 156, 164, 172, and sequences sharing at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NOs.4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108, 116, 124, 132,140, 148, 156, 164, 172, and/or a CDR3_(VH) sequence selected from thegroup consisting of SEQ ID NOs. 5, 13, 21, 29, 37, 45, 53, 61, 69, 77,85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173, and sequencessharing at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity to SEQ ID NOs. 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85,93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173.

In some embodiments, the vaccine-derived NAb has a CDR1_(VL) sequenceselected from the group consisting of SEQ ID NOs. 6, 14, 22, 30, 38, 46,54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142, 150, 158, 166,174, and sequences sharing at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to SEQ ID NOs. 6, 14, 22, 30, 38,46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134, 142, 150, 158, 166,174, a CDR2_(VL) sequence selected from the group consisting of SEQ IDNOs. 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127,135, 143, 151, 159, 167, 175, and sequences sharing at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to SEQID NOs. 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119,127, 135, 143, 151, 159, 167, 175, and/or a CDR3_(VL) sequence isselected from the group consisting of SEQ ID NOs. 8, 16, 24, 32, 40, 48,56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168,176, and sequences sharing at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to SEQ ID NOs. 8, 16, 24, 32, 40,48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168,176.

In a related aspect, the disclosure provided herein relates to acomposition for treating or preventing CMV infection comprising thevaccine-derived neutralizing antibody (NAb) against cytomegalovirus(CMV) described above. The composition may further comprise apharmaceutically acceptable carrier, excipient, diluent, adjuvant, orpreservative. Optionally, the NAb is a humanized antibody.

In another aspect, the disclosure provided herein relates to a smallpeptide comprising a linear epitope on UL128. In some embodiments, thesmall peptide comprises at least one cysteine residue. In someembodiments, the small peptide may have a size of 10 amino acids, 11amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 aminoacids, 16 amino acids, 17 amino acids, or 18 amino acids. In someembodiments, the small peptide comprises an amino acid sequence selectedfrom the group consisting of KRLDVCRAKMGYM (SEQ ID NO: 177),HKRLDVCRAKMGYM (SEQ ID NO: 178), KHKRLDVCRAKMGYM (SEQ ID NO: 179),non-native sequence KRLDVSRAKMGYMC (SEQ ID NO: 180), non-native sequenceKHKRLDVSRAKMGYMC (SEQ ID NO: 181), and a sequence which is at least 80%,at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to SEQ ID NOs: 177-181. Also disclosed herein is avaccine composition comprising one or more of the small peptides. Thevaccine composition may be used for treating or preventing CMVinfections. In certain embodiments, non-native sequence means that thesequence is artificial and is not found in nature.

In another aspect, the disclosure provided herein relates to a method ofproducing a vaccine-derived NAb against CMV. The method comprisesadministering to a subject an effective amount of a recombinant CMVpentameric complex comprising gH, gL, UL128, UL130 and UL131A, derivinghybridomas from the subject, and isolating NAbs from the hybridomas. Thesubject may be a mammal.

In another aspect, the disclosure provided herein relates to a method ofdetecting the presence of a CMV antigen in a biological sample or a cellculture comprising contacting the sample or the cell culture with avaccine-derived neutralizing antibody (NAb) against cytomegalovirus(CMV) described above.

In another aspect, the disclosure provided herein relates to a method oftreating or preventing CMV infection in a subject, comprisingadministering to the subject an effective amount of a compositioncomprising the vaccine-derived neutralizing antibody (NAb) againstcytomegalovirus (CMV) described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows EpC and FBs neutralizing antibody titers on serum samplesfrom immunized mice. Three Balb/C mice (X, RL and L) were vaccinatedthree times every four weeks by intraperitoneal (i.p.) route withMVA-gH/gL-PC. Mice were boosted with a fourth dose between week 48 and72 and spleens collected 4 days later for NAbs derivation. HCMV-specificserum NAb titer (IC50) was determined at different time pointspost-vaccination on EpC (ARPE-19) and FBs (MRC-5) using TB40/E.Arrowheads indicate vaccinations. Vaccinations were performed on all theanimals unless indicated under the arrowhead. Crosses represent the timeof spleen collection for the different animals. Dotted line indicatesthe detection limit of the assay.

FIG. 2A shows intracellular recognition of PC subunits by isolated NAb.Shown is FC staining by MVA-PC vaccine-derived NAb of permeabilizedBHK-21 cells infected with different MVA vectors expressing one or moresubunits of the PC.

FIG. 2B shows cell surface recognition of PC subunits by isolated NAb.Shown is FC staining by MVA-PC vaccine-derived NAb of non-permeabilizedBHK-21 cells infected with different MVA vectors expressing one or moresubunits of the PC. Uninfected cells and cells infected with an MVAencoding for a fluorescent protein (Venus) were used as negativecontrols.

FIG. 3 shows neutralization potency of vaccine-derived NAb. NAb derivedfrom MVA-PC immunized mice were used in a microneutralization assay todetermine the antibody concentrations required to prevent 50% infection(IC50) of ARPE-19 EpC, HUVEC EnC, and MRC-5 FB with HCMV strains TB40/Eand TR. CMV-HIG was used as a reference. Dotted line indicates thehighest antibody concentration used in the assay (25 μg/ml).

FIG. 4 shows inhibition of HCMV spread in EpC by NAb. ARPE-19 cells wereinfected with TB40/E or TR (MOI of 1), 24 hours later extensivelywashed, and incubated with serial dilutions of vaccine-derived NAb for 8days. Cells were imaged for GFP quantification. The graph shows the NAbconcentrations at which 50% reduction in the GFP positive area (IC50) incomparison to untreated controls was calculated. CMV-HIG was used as acontrol. The maximum evaluated antibody concentration is indicated bythe dotted line.

FIG. 5 shows NAbs mediated inhibition of TR and TB40/E cell-to-cellspread on EpCs. Shown are sequential pictures of TB40/E or TR infectedcells in the presence or absence of NAb 1B2, 21E9 and 62-11 added at 200μg/ml. NAbs were added at day 1 post-infection (P1) and pictures weretaken at the center of the wells at day 3, 6 and 8 PI. The samemagnification (50×) was used in all the pictures.

FIGS. 6A-6C show correlation analyses between NAb binding affinity andneutralizing potency. FIG. 6A shows NAb EC50 and EpC IC50 values thatwere plotted, and two-tailed Pearson analysis resulted in a positivecorrelation (r=0.7432, p=0.014). FIG. 6B shows NAb that were grouped inPC specific NAb (UL) and anti-gH NAb based on their subunit recognition(FIG. 2 and Table 2). No statistically significant difference betweenthe two groups was found using an unpaired t-test (p>0.05). FIG. 6Cshows that the same groups as in FIG. 6B were analyzed based on theirARPE-19 IC50 values. As evaluated using an unpaired t-test, neutralizingpotency of PC specific NAb was significantly different (p=0.0167) fromthat of gH NAb.

FIGS. 7A and 7B show recognition of linear gH by vaccine derivedgH-specific NAb. FIG. 7A shows immunoblot detection of gH expressed fromAd vectors in infected ARPE-19 EpC using vaccine-derived anti-gH NAb andanti-gH antibody AP86. Cells infected with Ad-tet were analyzed forcontrol. Shown is chemiluminescence detection of gH after short (5minutes) or long (1 hour) exposure of X-ray films to the immunoblot.MEK1/2 detection was performed as loading control. FIG. 7B showsimmunoblot detection of gH from HCMV strains Towne (TO), TR, Davis (DA),AD169 (AD) or TB40/E (TB) in infected FB using 18F10 and AP86.Uninfected cells (U) were used as a control. For control, samples wereanalyzed with anti-pp65 antibody (23-103). Mass markers (kDa) are shownnext to each panel.

FIGS. 8A and 8B show neutralization of CTB infection by vaccine-derivedNAb. FIG. 8A shows characterization of primary CTB. FSC vs. SSC dot ploton the left indicates the gated population of CTB analyzed. Histogramsrepresent cytokeratin 7 (center) and vimentin (right) expression of thegated CTB population. FIG. 8B shows results from NAb that were testedfor their ability to neutralize TB40/E infection of primary CTB isolatedfrom term placentae. Shown are the IC50 values for each vaccine-derivedNAb. CMV-HIG was used as a control. Dotted line indicates the highestantibody concentration used in the assay (50 μg/ml).

FIG. 9 shows an ELISA assay performed by coating the wells with L9L (2μg/mL), L10L (2 μg/mL) or PBS and incubating with anti-gH NAbs 18F10 orAP86 (2 μg/m L).

FIGS. 10A-10D show results of studies related to the mapping of the NAb13B5 minimal binding site. FIGS. 10A and 10B: C- and N terminaltruncated peptides based on library peptide 40 were used in an ELISA toidentify the shortest amino acid sequence needed for binding of NAb13B5. FIG. 10C: ELISA to compare 13B5 binding to K13M comprising theminimal 13B5 epitope sequence and peptides based on K13M with one (H14M)or two (K15M) additional amino acid residues of UL128 added to theN-terminus. FIG. 10D: Alanine scanning based on peptide K13M to identifyamino acid residues involved in 13B5 binding. Bars represent standarddeviation of triplicate wells.

FIGS. 11A-11C show binding and neutralizing antibody induction bypeptides based on the 13B5 epitope sequence. FIG. 11A: Peptides based onthe 13B5 binding site (K15M, K14CS and K16CS) and peptides containingonly partial sequences of the 13B5 binding site (UL128 library peptide38) were coupled to KLH and tested for binding to 13B5 antibody byELISA. KLH alone was used as a control. Bars represent standarddeviation of triplicate wells. FIGS. 11B and 11C: Balb/c mice (5 animalsper group) were immunized three times four weeks apart with KLH-coupledpeptides adjuvanted in Freund's adjuvant. FIG. 11B showspeptide-specific binding antibodies in sera of immunized mice weremeasured via ELISA 1 week before (−1wp1st) and 3 weeks post first,second, and third immunization (3wp1st; 3wp2nd, 3wp3rd) by using thepeptides as antigens that were used for the immunization. FIG. 11C showsserum neutralizing antibody titers (NT50) from immunized mice weremeasured against HCMV TB40/E on ARPE-19 EC using a standardmicroneutralization assay. Lines in B and C indicate the group mean.

DETAILED DESCRIPTION I. Vaccine-Derived NAbs

In one aspect, vaccine-derived neutralizing antibodies (NAbs) againstcytomegalovirus (CMV) are provided herein. Human cytomegalovirus (HCMV)elicits neutralizing antibodies (NAbs) of varying potency and cell-typespecificity to prevent HCMV entry into fibroblasts (FB) andepithelial/endothelial cells (EpC/EnC). NAbs targeting the majoressential envelope glycoprotein complexes gB and gH/gL inhibit both FBand EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnCis additionally blocked by extremely potent NAbs to conformationalepitopes of the gH/gL/UL128/UL130/UL131A pentamer complex (PC). Avaccine concept based on the delivery of a membrane tethered-PC byModified Vaccinia virus Ankara (MVA) (36), a widely used, clinical viralvector platform that has been safely tested in over 120,000 humans (51,52), was recently developed. This single vector, termed MVA-PC, canco-express all five PC subunits (the gH/gL/UL128/UL130/UL131A pentamercomplex, or “gH/gL-PC”). MVA-PC induced high titer and sustained NAbsagainst EpC/EnC entry in mice and rhesus monkeys and less potent NAbsthat blocked FB infection (36), which is consistent with the NAbresponses induced by HCMV during natural infection (37-39).

As provided herein, it was unexpectedly discovered that MVA-PC elicitsPC- and gH-specific NAbs having properties (such as cell-typespecificity, neutralization potency, minimal antigen recognition, andfrequency to recognize antigenic sites) similar or identical topreviously described NAbs isolated from human HCMV⁺ patients (32) orNAbs induced in a subject naturally infected with CMV. In addition,vaccine-derived PC-specific NAbs were shown to be significantly morepotent than gH-specific NAbs in preventing HCMV spread in EpC andinfection of primary cytotrophoblasts (CTBs) from term placentae,suggesting that NAbs recognizing the PC may play an important role ininterfering with HCMV vertical transmission.

As used herein, the term “vaccine-derived” antibody means that theantibody is produced by immunizing an animal using a vaccine in contrastto an antibody induced in a subject naturally infected with CMV. Forexample, an MVA vaccine for delivery of a UL128 complex and preventingCMV infection is described in PCT Publication No. WO 2014/018117 (“the'117 publication”), the content of which is incorporated by reference inits entirety. The vaccine described in the '117 publication, along withother recombinant complexes, may be used herein to immunize the animalfrom which the NAbs are derived. In certain embodiments, the animal maybe a mouse.

As used herein, the term “neutralizing antibody” or “neutralizingantibody against CMV” means that the antibody is capable of preventingor blocking CMV from infecting cells, such as epithelial cells,endothelial cells, primary placental cytotrophoblast cells, fibroblasts,cytotrophoblasts, or a combination thereof, in an animal, preferably ina mammal such as a human. The term can also mean that the antibody iscapable of preventing the spread of CMV in cell culture and neutralizingheterologous CMV strains.

The antibodies described herein may be monoclonal antibodies,recombinant antibodies or humanized antibodies.

The vaccine-derived NAbs disclosed herein may comprise a variable heavyregion comprising a CDR1_(VH) sequence, a CDR2_(VH) sequence, and aCDR3_(VH) sequence; and a variable light region comprising a CDR1_(VL)sequence, a CDR2_(VL) sequence, and a CDR3_(VL) sequence. Table 1 belowlists exemplary vaccine-derived NAbs and their sequences. Alsoencompassed herein are vaccine-derived NAbs comprising a variable heavyregion or a variable light region sharing at least 90% identity to thevariable heavy region or the variable light region of thevaccine-derived NAbs disclosed herein, or a combination thereof. Incertain embodiments, the vaccine-derived NAbs may comprise a variableheavy region or a variable light region sharing at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity to thevariable heavy region or the variable light region of thevaccine-derived NAbs disclosed herein or both.

TABLE 1 Sequences of Vaccine-Derived NAbs Nucleotide SequencesAmino Acid Sequences SEQ SEQ ID ID Description NO Sequence NO Sequence1B2-VH  1 gaagtgcagctggtggagtctgggggaggc  89 EVQLVESGGVLVKPttagtgaagcctggagggtccctgaaactct GGSLKLSCAASGFTFcctgtgcagcctctggattcactttcagtgact SDYYMYWVRQTPEKattacatgtattgggttcgccagactccggaa RLEWVATISDDGNYTaagaggctggagtgggtcgcaaccattagt NYPDSVKGRFTISRDgatgatggtaattacaccaactatccagaca NAKNNLYLQMSSLKSgtgtgaaggggcgattcaccatctccagag EDTAMYYCARGWLLacaatgccaagaacaatctttacctgcaaat PVFAYWGQGTLVTVgagcagtctgaagtctgaggacacagccat SA gtattactgtgcaagaggatggttactaccagtatttgcttactggggccaagggactctggtc actgtctctgctg 1B2-VL  2gatattgtgctaactcagtctccagccaccct  90 DIVLTQSPATLSVTPGgtctgtgactccaggagatagcgtcagtcttt DSVSLSCRASQSIGNcctgcagggccagccagagtattggcaaca NLHWYQQKSHESPRacctacactggtatcaacaaaaatcacatg LLIKYTSQSISGIPSRFagtctccaaggcttctcatcaaatatacttccc SGSGSGTDFTLNINSagtccatctctggaatcccctccaggttcagt VETEDFGVYFCQQSggcagtggatcagggacagatttcactctca NRWPWTFGGGTKLEatatcaacagtgtggagactgaagattttgg IK agtgtatttctgtcagcagagtaacagatggccgtggacgttcggtggaggcaccaagctgg aaatcaaac 1B2-VH-  3ggattcactttcagtgactatta  91 GFTFSDYY CDR1 1B2-VH-  4attagtgatgatggtaattacacc  92 ISDDGNYT CDR2 1B2-VH-  5gcaagaggatggttactaccagtatttgctta  93 ARGWLLPVFAY CDR3 ct 1B2-VL-  6cagagtattggcaacaac  94 QSIGNN CDR1 1B2-VL-  7 tatacttcc  95 YTS CDR21B2-VL-  8 cagcagagtaacagatggccgtggacg  96 QQSNRWPWT CDR3 54E11-VH  9cagatccagttggtgcagtctggacctgagc  97 QIQLVQSGPELKKPGtgaagaagcctggagagacagtcaagatct ETVKISCKASGYTFTcctgcaaggcttctggatataccttcacaagc SYGMNWVKQAPGKtatggaatgaactgggtgaagcaggctcca GLKWMGWINTYTGEggaaagggtttaaagtggatgggctggata PTYADDFKGRFAFSLaacacctacactggagagccaacatatgct ETSASTAYLQINNLKgatgacttcaagggacggtttgccttctctttg NEDTATYFCAREHYYgaaacctctgccagcactgcctatttacagat GINPLLGCWGQGTTLcaacaacctcaaaaatgaggacacggcta TVSS catatttctgtgcaagagaacattactacggtattaacccccttttaggctgctggggccaagg caccactctcacagtctcctcag 54E11-VL 10gatatccagatgacacagactacatcctccc  98 DIQMTQTTSSLSASLtgtctgcctctctgggagacagagtcaccatc GDRVTISCSASQGISagttgcagtgcaagtcagggcattagcaatt NYLNWYQQKPDGTVatttaaactggtatcagcagaaaccagatgg KLLIYDTSSLHSGVPSaactgttaaactcctgatctatgacacatcaa RFSGSGSGTDYSLTIgtttacactcaggagtcccatcaaggttcagt SNLEPEDIATYYCQQggcagtgggtctgggacagattattctctcac YSKLPYTFGGGTKLEaatcagcaacctggaacctgaagatattgc IK cacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctgga aataaaac 54E11-VH- 11ggatataccttcacaagctatgga  99 GYTFTSYG CDR1 54E11-VH- 12ataaacacctacactggagagcca 100 INTYTGEP CDR2 54E11-VH- 13gcaagagaacattactacggtattaaccccc 101 AREHYYGINPLLGC CDR3 ttttaggctgc54E11-VL- 14 cagggcattagcaattat 102 QGISNY CDR1 54E11-VL- 15 gacacatca103 DTS CDR2 54E11-VL- 16 cagcagtatagtaagcttccttacacg 104 QQYSKLPYT CDR321F6-VH 17 cagatccagttggtgcagtctggacctgagc 105 QIQLVQSGPELKKPGtgaagaagcctggagagacagtcaagatct ETVKISCKASGYTFTcctgcaaggcttctggatataccttcacaagc SYGMNWVKQAPGKtatggaatgaactgggtgaagcaggctcca GLKWMGWINTYTGEggaaagggtttaaagtggatgggctggata PTYADDFKGRFAFSLaacacctacactggagagccaacatatgct ETSASTAYLQINNLKgatgacttcaagggacggtttgccttctctttg NEDTATYFCAREHYYgaaacctctgccagcactgcctatttacagat GINPLLGCWGQGTTLcaacaacctcaaaaatgaggacacggcta TVSS catatttctgtgcaagagaacattactacggtattaacccccttttaggctgctggggccaagg caccactctcacagtctcctcag 21F6-VL 18gatatccagatgacacagactacatcctccc 106 DIQMTQTTSSLSASLtgtctgcctctctgggagacagagtcaccatc GDRVTISCSASQGISagttgcagtgcaagtcagggcattagcaatt NYLNWYQQKPDGTVatttaaactggtatcagcagaaaccagatgg KLLIYDTSSLHSGVPSaactgttaaactcctgatctatgacacatcaa RFSGSGSGTDYSLTIgtttacactcaggagtcccatcaaggttcagt SNLEPEDIATYYCQQggcagtgggtctgggacagattattctctcac YSKLPYTFGGGTKLEaatcagcaacctggaacctgaagatattgc IK cacttactattgtcagcagtatagtaagcttccttacacgttcggaggggggaccaagctgga aataaaac 21F6-VH- 19ggatataccttcacaagctatgga 107 GYTFTSYG CDR1 21F6-VH- 20ataaacacctacactggagagcca 108 INTYTGEP CDR2 21F6-VH- 21gcaagagaacattactacggtattaaccccc 109 AREHYYGINPLLGC CDR3 ttttaggctgc21F6-VL- 22 cagggcattagcaattat 110 QGISNY CDR1 21F6-VL- 23 gacacatca 111DTS CDR2 21F6-VL- 24 cagcagtatagtaagcttccttacacg 112 QQYSKLPYT CDR312E2-VH 25 gaagtgaagctggtggagtctgggggagg 113 EVKLVESGGGLVQPcttagtgcagcctggagggtccctgaaactct GGSLKLSCATSGFTFcctgtgcaacctctggattcactttcagtgact SDYYMFWVRQTPEKattacatgttttgggttcgccagactccagag KLEWVAYISNGGGSTaagaagctggagtgggtcgcatacattagta YYPDTVKGRFTISRDatggtggtggtagcacctattatccagacact NDKNTLYLQMSRLKSgtaaagggccgattcaccatctccagagac DDTALYYCVRPKRDFaatgacaagaacaccctatacctgcaaatg QYLYAMDYWGQGTSagtcgtctgaagtctgacgacacagccttgt VTVSS attactgtgtaagaccgaaacgggactttcaatacctctatgctatggactactggggtcaag gaacctcagtcaccgtctcctcag 12E2-VL 26gacattgtgctgacacagtctcctgcttcctta 114 DIVLTQSPASLAVSLgctgtatctctggggcagagggccaccatct GQRATISCRASKSVScatgcagggccagcaaaagtgtcagtacat TSGYSYMHWYQQKPctggctatagttatatgcactggtaccaacag GQSPKLLIYLASNLESaaaccaggacagtcacccaaactcctcatc GVPARFSGSGSGTDtatcttgcatccaacctagaatctggggtccct FTLNIHPVEDEDAATgccaggttcagtggcagtgggtctgggaca YYCQHSRELPWTFGgacttcaccctcaacatccatcctgtggagg GGTKLEIK acgaggatgctgcaacctattactgtcagcacagtagggagcttccgtggacgttcggtgga ggcaccaagctggaaatcaaac 12E2-VH- 27ggattcactttcagtgactattac 115 GFTFSDYY CDR1 12E2-VH- 28attagtaatggtggtggtagcacc 116 ISNGGGST CDR2 12E2-VH- 29gtaagaccgaaacgggactttcaatacctct 117 VRPKRDFQYLYAMD CDR3 atgctatggactac Y12E2-VL- 30 aaaagtgtcagtacatctggctatagttat 118 KSVSTSGYSY CDR1 12E2-VL-31 cttgcatcc 119 LAS CDR2 12E2-VL- 32 cagcacagtagggagcttccgtggacg 120QHSRELPWT CDR3 13B5-VH 33 caggttactctgaaagagtctggccctggga 121QVTLKESGPGILKPS tattgaagccctcacagaccctcagtctgact QTLSLTCSFSGFSLTtgttctttctctgggttttcactgaccacttctggtt TSGLGVGWIRQPSGtgggtgtaggctggattcgtcagccttcaggg KGLEWLAHIWWDDDaagggtctggagtggctggcacacatttggt KYFNPSLRNQLTISKgggatgatgataaatactttaacccatccctg DTSRNQVFLEITSVTTaggaaccagctcacaatctccaaggatacc ADTATYYCVRSLYDYtccagaaaccaggtattcctcgagatcacca DEGYYFDSWGQGTTgtgtgaccactgcagatactgccacttactac LTVSS tgtgttcgaagcctttatgattacgacgaggggtactactttgactcctggggccaaggcacc actctcacagtctcctcag 13B5-VL 34gacattgtgatgactcagtctccagccaccct 122 EIVMIQSPATLSVNPGgtctgtgaatccaggagatagagtctctctct DRVSLSCRASQSISDcctgcagggccagccagagtattagcgact YLHWYQQKSHESPRacttacactggtatcaacaaaaatcacatga LLIKYASQSISGIPSRgtctccaaggcttctcatcaaatacgcttccc FSGSGSGSDFTLSINaatccatctctgggatcccctccaggttcagt SVEPEDVGVYYCQNggcagtggatcagggtcagatttcactctca GHTFPPTFGGGTKLEgtatcaacagtgtggaacctgaagatgttgg IK agtgtattattgtcaaaatggtcacacctttcctccgacgttcggtggaggcaccaagctgga aatcaaac 13B5-VH- 35gggttttcactgaccacttctggtttgggt 123 GFSLTTSGLG CDR1 13B5-VH- 36atttggtgggatgatgataaa 124 IWWDDDK CDR2 13B5-VH- 37gttcgaagcctttatgattacgacgaggggta 125 VRSLYDYDEGYYFDS CDR3 ctactttgactcc13B5-VL- 38 cagagtattagcgactac 126 QSISDY CDR1 13B5-VL- 39 tacgcttcc 127YAS CDR2 13B5-VL- 40 caaaatggtcacacctttcctccgacg 128 QNGHTFPPT CDR318F10-VH 41 cagattactcagaaagagtctggccctggg 129 QVTLKESGPGILQPSatattgcagccctcccagaccctcagtctgac QTLSLTCSFSGFSLSttgttctttctctgggttttcactgagcacttatgg TYGIGIGWIRQPSGKtataggaataggctggattcgtcagccttcag GLEWLAHIWWNDNKggaagggtctggagtggctggcacacatttg NYNTALKSRLTISKDPgtggaatgataataagaactataacacagc SNNQVFLKIASVDTAcctgaagagccggctcacaatctccaagga DTATYFCARTGYFDVtccctccaacaaccaggtattcctcaagatc WGAGTTVTVSSgccagtgtggacactgcagatactgccacat acttctgtgctcgaactgggtacttcgatgtctggggcgcagggaccacggtcaccgtctcct cag 18F10-VL 42gatgttgtgatgacccaaactccactctccct 130 DVVLTQTPLSLPVSLgcctgtcagtcttggagatcaagtctccatttct GDQVSISCSSSQSLVtgcagctctagtcagagccttgtgcacagta HSNGNTYIHWYLQKPatggaaacacctatatacattggtacctgca GQSPKLLIYTVSNRFgaaaccaggccagtctccaaagctcctgat SGVPDRFSGSGSGTctacacagtttccaaccgattttctggggtccc DFTLKISRVEAEDLGLagacaggttcagtggcagtggatcagggac YFCSQSTHVPYTFGagatttcacactcaagatcagcagagtgga GGTKLEIKggctgaggatctgggactttatttctgctctca aagtacacatgttccgtacacgttcggaggggggaccaagctggaaataaaac 18F10-VH- 43 gggttttcactgagcacttatggtatagga 131GFSLSTYGIG CDR1 18F10-VH- 44 atttggtggaatgataataag 132 IWWNDNK CDR218F10-VH- 45 gctcgaactgggtacttcgatgtc 133 ARTGYFDV CDR3 18F10-VL- 46cagagccttgtgcacagtaatggaaacacc 134 QSLVHSNGNTY CDR1 tat 18F10-VL- 47acagtttcc 135 TVS CDR2 18F10-VL- 48 tctcaaagtacacatgttccgtacacg 136SQSTHVPYT CDR3 21E9-VH 49 cagatccagttggtgcagtctggacctgagc 137QIQLVQSGPELKKPG tgaagaagcctggagagacagtcaagatct ETVKISCKASGYTFTIcctgcaaggcttctgggtataccttcacaatct YGMNWVKQAPGKGLatggaatgaactgggtgaagcaggctccag KWMGWINTYTGEPTgaaagggtttaaagtggatgggctggataa YADDFRGRFAFSLETacacctacactggagagccaacatatgctg SASTAYLQINNLKNEatgacttcaggggacggtttgccttctctttgg DTATYFCARKGYYGaaacctctgccagcactgcctatttgcagatc SSGYFDYWGQGTTLaacaacctcaaaaatgaggacacggctac TVSS atatttctgtgcaagaaaggggtactacggtagtagcgggtactttgactactggggccaagg caccactctcacagtctcctcag 21E9-VL 50agtattgtgatgacccagactcccaaattcct 138 SIVMTQTPKFLLVSAgcttgtatcagcaggagacagggttaccata GDRVTITCKASQSVSacctgcaaggccagtcagagtgtgagtaat NDVSWYQQKPGQSPgatgtatcttggtaccaacagaagccaggg KLLIYYASNRYTGVPcagtctcctaaactgctgatatactatgcgtcc DRFTGSGYGTDFTFTaatcgctacactggagtccctgatcgcttcac ISTVQAEDLAVYFCQtggcagtggatatgggacggatttcactttca QDYSSPWTFGGGTKccatcagcactgtgcaggctgaagacctgg LEIK cagtttatttctgtcagcaggattatagctctccgtggacgttcggtggaggcaccaagctgga aatcaaac 21E9-VH- 51gggtataccttcacaatctatgga 139 GYTFTIYG CDR1 21E9-VH- 52ataaacacctacactggagagcca 140 INTYTGEP CDR2 21E9-VH- 53gcaagaaaggggtactacggtagtagcgg 141 ARKGYYGSSGYFDY CDR3 gtactttgactac21E9-VL- 54 cagagtgtgagtaatgat 142 QSVSND CDR1 21E9-VL- 55 tatgcgtcc 143YAS CDR2 21E9-VL- 56 cagcaggattatagctctccgtggacg 144 QQDYSSPWT CDR34A3-VH 57 caggtccaactgcagcagcctggggctgag 145 QVQLQQPGPELVRPctggtgaggcctggggcttcagtgaaactgt GASVKLSCKASGYTFcctgcaaggcttctggctacaccttcaccatct TIYWMNWVKQRPGQactggatgaactgggtgaagcagaggcctg GLEWIGMIDPSDSETgacaaggccttgaatggattggtatgattgat HYNQMFKDKATLTVccttcagacagtgaaactcactacaatcaga DKSSSTAYMQLSSLTtgttcaaggacaaggccacattgactgtaga SEDSAVYYCASSGTcaaatcctccagcactgcctacatgcagctc GAYWGQGTLLTVSAagcagcctgacatctgaggactctgcggtct attactgtgcaagttctgggacgggggcttactggggccaagggactctgctcactgtctctgc ag 4A3-VL 58gatgttgtgatgacccagactccactcactttg 146 DVVMTQTPLTLSVTItcggttaccattggacaaccagcctccatctc GQPASISCKSSQSLLttgcaagtcaagtcagagcctcttagatagtg DSDGKTYLNWLLQRatggaaagacatatttgaattggttgttacag PGQSPKRLIYLVSKLaggccaggccagtctccaaagcgcctgatc DSGVPDRFTGSGSGtatttggtgtctaaactggactctggagtccct TDFTLKISRLEAEDLGgacaggttcactggcagtggatcagggaca VYYCWQGTHFPYTFgatttcacactgaaaatcagcagattggaag GGGTKLEIKctgaggatttgggagtttattattgctggcaag gtacacattttccgtacacgttcggaggggggaccaagctggaaataaaac 4A3-VH- 59 ggctacaccttcaccatctactgg 147 GYTFTIYWCDR1 4A3-VH- 60 attgatccttcagacagtgaaact 148 IDPSDSET CDR2 4A3-VH- 61gcaagttctgggacgggggcttac 149 ASSGTGAY CDR3 4A3-VL- 62cagagcctcttagatagtgatggaaagacat 150 QSLLDSDGKTY CDR1 at 4A3-VL- 63ttggtgtct 151 LVS CDR2 4A3-VL- 64 tggcaaggtacacattttccgtacacg 152WQGTHFPYT CDR3 62-11-VH 65 caggtccaactgcagcagcctggggctgag 153QVQLQQPGAELVRP ctggtgaggcctggggcttcagtgaagctgt GASVKLSCKASGYTFcctgcaaggcttctggctacaccttcaccagc TSYWMNWVKQRPGtactggatgaactgggtgaagcagaggcct QGLEWIGMIDPSDSEggacaaggccttgaatggattggtatgattg THYNQMFKDKATLTVatccttcagacagtgaaactcactacaatca DKSSSTAYMQLSSLTaatgttcaaggacaaggccacattgactgta SEDSAVYYCSNGYSgacaaatcctccagcacagcctacatgcaa SFAYWGQGTLVTVSctcagcagcctgacatctgaggactctgcgg V tctattactgttcaaatggttactcctcctttgcttactggggccaagggactctggtcactgtctct gtag 62-11-VL 66gatgtccagatgacacagactacatcctccc 154 DVQMTQTTSSLSASLtgtctgcctctctgggagacagagtcaccatc GDRVTISCSASQGISagttgcagtgcaagtcagggcattagcaatt NYLNWYQQKPDGTVatttaaactggtatcagcagaaaccagatgg KLLIYDTSSLHSGVPSaactgttaaactcctgatctatgacacatcaa RFSGSGSGTDYSLTIgtttacactcaggagtcccatcaaggttcagt SNLEPEDIATYYCQQggcagtgggtctgggacagattattctctcac YSKLPYTFGGGTKLEaatcagcaacctggaacctgaagatattgc IK cacttactattgtcagcagtatagtaagcttccctacacgttcggaggggggaccaagctgg aaataaaac 62-11-VH- 67ggctacaccttcaccagctactgg 155 GYTFTSYW CDR1 62-11-VH- 68attgatccttcagacagtgaaact 156 IDPSDSET CDR2 62-11-VH- 69tcaaatggttactcctcctttgcttac 157 SNGYSSFAY CDR3 62-11-VL- 70cagggcattagcaattat 158 QGISNY CDR1 62-11-VL- 71 gacacatca 159 DTS CDR262-11-VL- 72 cagcagtatagtaagcttccctacacg 160 QQYSKLPYT CDR3 62-100-VH 73caggtccaactgcagcagcctggggctgag 161 QVQLQQPGAELVRPctggtgaggcctggggcttcagtgaagctgt GASVKLSCKASGYTFcctgcaaggcttctggctacaccttcaccagc TSYWMNWVKQRPGtactggatgaactgggtgaagcagaggcct QGLEWIGMIDPSDSEggacaaggccttgaatggattggtatgattg THYNQMFKDKATLTVatccttcagacagtgaaactcactacaatca DKSSSTAYMQLSSLTaatgttcaaggacaaggccacattgactgta SEDSAVYYCSNGYSgacaaatcctccagcacagcctacatgcaa SFAYWGQGTLVTVSctcagcagcctgacatctgaggactctgcgg V tctattactgttcaaatggttactcctcctttgcttactggggccaagggactctggtcactgtctct gtag 62-100-VL 74gatattgtgctaactcagtctccagccaccct 162 DIVLTQSPATLSVTPGgtctgtgactccaggagatagcgtcagtcttt DSVSLSCRASQSISNcctgcagggccagccaaagtattagcaaca NLHWYQQKSHESPRacctacactggtatcaacaaaaatcacatg LLIKYASQSISGIPSRagtctccaaggcttctcatcaagtatgcttccc FSGSGSGTDFTLSINagtccatctctgggatcccctccaggttcagt SVETEDFGKYVCQQggcagtggatcagggacagatttcactctca SNSWPLTFGSGTKLEgtatcaacagtgtggagactgaagattttgg IK aaagtatgtctgtcaacagagtaacagctggccactcacgttcggctcggggacaaagttgg aaataaaac 62-100-VH- 75ggctacaccttcaccagctactgg 163 GYTFTSYW CDR1 62-100-VH- 76attgatccttcagacagtgaaact 164 IDPSDSET CDR2 62-100-VH- 77tcaaatggttactcctcctttgcttac 165 SNGYSSFAY CDR3 62-100-VL- 78caaagtattagcaacaac 166 QSISNN CDR1 62-100-VL- 79 tatgcttcc 167 YAS CDR262-100-VL- 80 caacagagtaacagctggccactcacg 168 QQSNSWPLT CDR3 2-80-VH 81cagatccagttggtgcagtctggacctgagc 169 QIQLVQSGPELKKPGtgaagaagcctggagagacagtcaagatct ETVKISCKASGYTFTcctgcaaggcttctggatataccttcacaaac NFGMNWVKQAPGKtttggaatgaactgggtgaagcaggctccag GLKWMGWINTYTGEgaaagggtttaaagtggatgggctggataa PTYADDFKGRFAFSLacacctacactggagagccaacatatgctg ETSASTASLQINNLKatgacttcaagggacggtttgccttctctttgg NEDTATYFCARRGDaaacctctgccagcactgcctctttgcagatc GLYSMDYWGQGTSVaacaacctcaaaaatgaggacacggctac TVSS atatttctgtgcaagaaggggggatggcctctattctatggactactggggtcaaggaacctc agtcaccgtctcctcag 2-80-VL 82gacattgtgctgacccaatctccagcttctttg 170 DIVLTQSPASLAVSLgctgtgtctctggggcagagggccaccatat GQRATISCRASESIDcctgcagagccagtgaaagtattgatagttat SYGNSFMYWYQQKPggcaatagttttatgtactggtaccagcagaa GQPPKLLIYRASNLEaccaggacagccacccaaactcctcatctat SGIPARFSGSGSRTDcgtgcatccaacctagaatctgggatccctg FTLTINPVEADDVATYccaggttcagtggcagtgggtctaggacag YCQQSNEDPLTFGAacttcaccctcaccattaatcctgtggaggct GTKLELKgatgatgttgcaacctattactgtcagcaaag taatgaggatcctctcacgttcggtgctgggaccaagctggagctgaaac 2-80-VH- 83 ggatataccttcacaaactttgga 171 GYTFTNFGCDR1 2-80-VH- 84 ataaacacctacactggagagcca 172 INTYTGEP CDR2 2-80-VH- 85gcaagaaggggggatggcctctattctatgg 173 ARRGDGLYSMDY CDR3 actac 2-80-VL- 86gaaagtattgatagttatggcaatagtttt 174 ESIDSYGNSF CDR1 2-80-VL- 87 cgtgcatcc175 RAS CDR2 2-80-VL- 88 cagcaaagtaatgaggatcctctcacg 176 QQSNEDPLT CDR3

In some embodiments, the vaccine-derived NAbs disclosed herein such as1B2, 54E11, 21F6, 12E2, 1365 and 4A3 are potent NAbs that are able toneutralize HCMV infection of epithelial cells, endothelial cells andprimary placental cytotrophoblast cells at picomolar concentrations.These NAbs also prevent the spread of the virus in cell culture andneutralize heterologous HCMV strains with the same potency. SomePC-specific NAbs, such as 1B2, 54E11, 21F6, 12E2, and 4A3, specificallybind to conformational epitopes composed of UL128/UL130/UL131A orUL130/UL131A subunits of the gH/gL-PC. Some PC-specific NAbs such as13B5 bind a linear epitope on UL128.

In other embodiments, the NAbs disclosed herein such as 21E9, 62-11,62-100, 2-80, 1361, 6G2, 10G6, and 25H10 specifically bind toconformational epitopes on gH and prevent HCMV infection of fibroblasts,epithelial cells, endothelial cells and cytotrophoblasts with similarpotency, even if less efficiently that the PC-specific NAbs targetingthe UL subunits. Some NAbs, such as 18F10, bind to a linear epitope ongH and neutralize CMV infection of epithelial cells but not CMVinfection of fibroblasts with potency comparable to other gH NAbs. Allthe NAbs tested showed binding affinity for the gH/gL-PC in the highnanomolar to low picomolar range.

In some embodiments, the vaccine-derived NAbs disclosed herein potentlyinterfere with HCMV cell-to-cell spread and/or syncytia formation inEpC. Previous reports have shown that CMV-HIG, which represents a pooledIgG antibody repertoire from over 1,000 HCMV⁺ individuals, potentlyprevents EpC spread of different HCMV strains (72). In contrast, Jacobet al. have shown that CMV-HIG and monoclonal NAb targeting gB, gH, orthe PC are unable to prevent HCMV spread in EpC. However, Jacob et al.investigated spread inhibition in the presence of only very low antibodyconcentration (76). As disclosed herein, about 1,000-fold higher amountsof the vaccine-derived NAbs than that used by the prior art were shownto be effective in preventing HCMV cell-to-cell spread and/or syncytiaformation in EpC of heterologous HCMV strains (Table 3). In addition,the vaccine-derived PC-specific NAbs disclosed herein were significantlymore potent than the anti-gH NAbs disclosed herein or CMV-HIG tointerfere with EpC spreading of HCMV. Hence, PC-specific NAbs induced byMVA-PC not only confer potent inhibition of HCMV entry, but also havepotent ability to prevent HCMV spread and/or syncytia formation in EpC,suggesting that the anti-PC NAbs elicited by MVA-PC can limitcell-associated virus dissemination throughout the human host andtransmission to the fetus.

In some embodiments, the vaccine-derived NAbs disclosed hereindemonstrate a positive correlation between antibody neutralizing potencyand binding affinity of PC- and gH-specific NAbs recognizing cellsurface PC. However, in contrast to the difference in neutralizationpotency of the vaccine-derived PC-specific NAbs and anti-gH NAbsdisclosed herein, the difference in binding affinity between these twogroups of NAbs was not significant. It is possible that the significantdifference in neutralization potency between PC-specific NAbs and NAbstargeting gH/gL (or gB) may reflect the relative low amount of theUL128/130/131A subunits in HCMV virions compared to gH/gL. Hence, muchlower antibody concentrations are required to interfere with PC-mediatedentry than with the fusion function of gH/gL (77). In contrast, thedifference in neutralizing potency of individual NAbs targeting theUL128/130/131A subunits of the PC may be a function of their bindingaffinity.

II. Small Peptides Comprising Epitopes for Vaccine-Derived NAbs

Two of the isolated NAbs, 13B5 and 18F10, showed recognition of linearepitopes on UL128 and gH respectively. No linear epitope on UL128 withneutralizing properties has been described in the prior art, thus thesmall peptides disclosed herein can represent the epitope as a surrogatefor the whole gH/gL-PC in a peptide vaccine setting. In someembodiments, the small peptide comprises at least one cysteine residuesuch that a disulfide bridge between UL128 and gL can be formed. In someembodiments, the small peptide may have a size of 10 amino acids, 11amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 aminoacids, 16 amino acids, 17 amino acids or 18 amino acids. Other examplesinclude non-native derivatives of SEQ ID NO: 177, e.g. SEQ ID NOs: 180and 181.

By using 13B5 to bind to a UL128 peptide library in an ELISA assay,peptide K13M (KRLDVCRAKMGYM) (SEQ ID NO: 177) was identified as theminimum sequence required to have a strong binding of the NAb (FIGS.10A-10B). Moreover, the longer peptides H14M (HKRLDVCRAKMGYM) (SEQ IDNO: 178) and K15M (KHKRLDVCRAKMGYM) (SEQ ID NO: 179) were efficientlyrecognized by 13B5 (FIG. 10C). The cysteine (Cys-162) residue centrallylocated in the recognized peptides has been indicated as the amino acidresponsible for the disulfide bridge between UL128 and gL thusconnecting gH/gL to the UL subunits. The involvement of Cys-162 in 13B5binding to K13M was evaluated. When a new peptide in which Cys-162 wassubstituted with serine (K13MS, KRLDVSRAKMGYM) (SEQ ID NO: 182) wastested, 13B5 binding was not detected. When Cys was added to the end ofpeptide K13MS (i.e., K14CS, KRLDVSRAKMGYMC) (SEQ ID NO: 180)), 13B5binding was completely restored. It was unexpected that the addition ofa cysteine to the 3′ end of peptide K13MS (SEQ ID NO: 182) to formpeptide K14CS (i.e., SEQ ID NO: 180) would completely restore binding to13B5 since no binding was detected with K13MS. Similarly, 13B5 bindingis detected when Cys was added to the end of K15M and the Cys-162substituted with serine (i.e., K16CS, KHKRLDVSRAKMGYMC) (SEQ ID NO:181),

The small peptides comprising one or more linear epitopes on thegH/gL-PC disclosed herein can be used as peptide vaccines. In someembodiments, K15M (KHKRLDVCRAKMGYM, SEQ ID NO: 179), K14CS(KRLDVSRAKMGYMC, SEQ ID NO: 180), and K16CS (KHKRLDVSRAKMGYMC, SEQ IDNO:181) can be used as peptide vaccines. Upon administration to asubject, a peptide vaccine stimulates antibody production that isspecific for CMV antigens on the surface of virus-infected cells orother cells that have incorporated the protein through variousprocessing mechanisms. A peptide vaccine uses one or more small peptidesas antigens to elicit an immune response. In order to induce PC specificNAbs the peptides can be coupled to a carrier protein such as KeyholeLimpet Hemocyanin (KLH), Tetanus and diphtheria toxoids (TT and DTrespectively), Hepatitis B surface antigen (HBsAg). For example, in someembodiments, KLH-K15M (KLH-coupled SEQ ID NO: 179), KLH-K14CS(KLH-coupled SEQ ID NO: 180), or KLH-K16CS (KLH-coupled SEQ ID NO:181)can be used to elicit an immune response. Given the peptides lowimmunogenicity in vivo, adjuvants can be added. The peptide vaccine mayfurther comprise one or more adjuvants to boost the immune response.Possible adjuvants include Freud's Complete and Incomplete (CFA, IFA),squalene-based oil-in-water nano-emulsions (MF59, AddaVax), aluminumhydroxide suspensions (Alum, Alhydrogel), toll like receptor agonists(monophosphoryl lipid A), pathogen associated molecular pattern (PAMP)agonists, and damage associated molecular pattern (DAMP) agonists. Otheradjuvants in use are so-called oil-in-water emulsions, saponin, LPS,quit-A, Montanide, RIBI, and others that are known in the field.Emulsification of a peptide or peptides in the adjuvant is used as asubcutaneous injection that has benefits for protection againstpathogens both in humans and in veterinary applications.

It is desirable to develop effective peptide vaccines due to the easeand low cost for synthesizing small peptides, the effectiveness of thepeptide vaccines in inducing immune response, and improved clinicalsafety in general. Peptides are synthetic molecules which are not liveor propagating and have an overwhelming safety advantage. They can beproduced using standard clinical manufacturing techniques to highprecision and purity. They can be freeze-dried and transported easilyand in that condition, avoid the necessity for cold chain. In regards totheir specificity, because of the short sequence that defines thepeptide vaccine disclosed herein, it has inherent specificity in regardsto having limited sequence for the immune system to recognize andprocess to generate off-target antibodies or T cell responses. Othersalutary benefits of peptide vaccines include the possibility ofdeveloping a multi-valent formula that is specific for differentantigens or different key locations in an antigen that can cause thedevelopment of unique and non-overlapping neutralizing antibodies thatcan aid in protection against a pathogen.

In some embodiments, the small peptides can be administered to asubject, either alone or in combination with one or more adjuvants, toelicit an immune response against CMV infection of the subject. In someembodiments, the small peptides can be administered to a subject such asa mammal to produce NAbs against CMV. In certain embodiments, a methodof producing NAbs against CMV may include administering a first dose ofone or more small peptides to a subject and administering a second doseof one or more small peptides to the subject after administration of thefirst dose. In certain embodiments, the second dose may be administeredto the subject about one week, about two weeks, about three weeks, aboutfour weeks, or about five weeks after administration of the first dose.In certain embodiments, at least one of the first and second dosesincludes administering one or more small peptides, at least one of whichis selected from SEQ ID NOs: 179, 180, and 181. In certain embodiments,the first dose includes administering one or more small peptides, atleast one of which is selected from SEQ ID NOs: 179, 180, and 181. Incertain embodiments, the first dose includes administering SEQ IDNO:179, SEQ ID NO: 180, or SEQ ID NO:181 and the second dose includesadministering SEQ ID NO:179, SEQ ID NO: 180, or SEQ ID NO:181. Incertain embodiments, the first and second doses include administeringSEQ ID NO:179, SEQ ID NO: 180, or SEQ ID NO:181. Any combination of SEQID NOS 179, 180, and 181 may be used for the first and second doses. Forexample, the first dose and the second dose comprise at least one of SEQID NOS 179, 180, or 181, as shown in the table below:

First Dose comprises: Second Dose comprises: SEQ ID NO: 179* SEQ ID NO:179**** SEQ ID NO: 179* SEQ ID NO: 180***** SEQ ID NO: 179* SEQ ID NO:181****** SEQ ID NO: 180** SEQ ID NO: 179**** SEQ ID NO: 180** SEQ IDNO: 180***** SEQ ID NO: 180** SEQ ID NO: 181****** SEQ ID NO: 181*** SEQID NO: 179**** SEQ ID NO: 181*** SEQ ID NO: 180***** SEQ ID NO: 181***SEQ ID NO: 181****** *The first dose may also include SEQ ID NO: 180and/or SEQ ID NO: 181 **The first dose may also include SEQ ID NO: 179and/or SEQ ID NO: 181 ***The first dose may also include SEQ ID NO: 179and/or SEQ ID NO: 180 ****The second dose may also include SEQ ID NO:180 and/or SEQ ID NO: 181 *****The second dose may also include SEQ IDNO: 179 and/or SEQ ID NO: 181 ******The second dose may also include SEQID NO: 179 and/or SEQ ID NO: 180

The NAbs can be used as a therapeutic agent against CMV infection, asdiscussed above.

III. Therapeutic and Vaccine Compositions

Based on the vaccine-derived NAbs and the small peptides describedabove, therapeutic or vaccine compositions for treating or preventingCMV infection are also provided. In some embodiments, a therapeuticcomposition may include one or more vaccine-derived NAbs describedabove. In certain embodiments, a vaccine composition may include one ormore small peptides comprising one or more linear epitopes on thegH/gL-PC. The one or more linear epitopes may be any of the epitopesdescribed above. In such embodiments, the vaccine composition can be amultivalent vaccine comprising two or more small peptides, each peptidecomprising a different linear epitope. The linear epitopes may bederived from (i.e., the epitope sequence is part of) the same subunit ordifferent subunits of the gH/gL-PC. In one aspect, a multivalent vaccinemay comprise two or more small peptides, each of which comprise adifferent linear epitope on a single subunit of the gH/gL-PC. Forexample, the two or more linear epitopes may be derived from the UL128subunit of the gH/gL-PC; the two or more linear epitopes may be derivedfrom the gH subunit of the gH/gL-PC, the two or more linear epitopes maybe derived from the gL subunit of the gH/gL-PC, the two or more linearepitopes may be derived from the UL130 subunit of the gH/gL-PC; or thetwo or more linear epitopes may be derived from the UL131A subunit ofthe gH/gL-PC. In another aspect, a multivalent vaccine may comprise twoor more small peptides, wherein the two or more small peptides comprisedifferent linear epitopes derived from two or more different subunits ofthe gH/gL-PC. For example, the multivalent vaccine may comprise two ormore small peptides, each of which comprises one or more linearepitopes, wherein at least one of the linear epitopes on one of thesmall peptides is derived from the UL128 subunit of the gH/gL-PC and atleast one of the linear epitopes on another small peptide is derivedfrom the gH subunit of the gH/gL-PC. Such therapeutic or vaccinecompositions can be administered to a subject to treat or prevent CMVinfections, particularly HCMV infections.

The therapeutic or vaccine compositions described above may also includeone or more pharmaceutically acceptable carrier. A “pharmaceuticallyacceptable carrier” refers to a pharmaceutically acceptable material,composition, or vehicle that is involved in carrying or transporting acompound of interest from one tissue, organ, or portion of the body toanother tissue, organ, or portion of the body. For example, the carriermay be a liquid or solid filler, diluent, excipient, solvent, orencapsulating material, or some combination thereof. Each component ofthe carrier must be “pharmaceutically acceptable” in that it must becompatible with the other ingredients of the formulation. It also mustbe suitable for contact with any tissue, organ, or portion of the bodythat it may encounter, meaning that it must not carry a risk oftoxicity, irritation, allergic response, immunogenicity, or any othercomplication that excessively outweighs its therapeutic benefits.

IV. Methods of Treating or Preventing CMV Infections

The therapeutic or vaccine compositions described herein may be used totreat or prevent any HCMV infection that infects epithelial cells,endothelial cells, fibroblasts or a combination thereof. Examples ofHCMV infections that may be treated or prevented using the methodsdescribed herein may include, but is not limited to, congenital HCMVinfection, opportunistic HCMV infections in subjects with compromisedimmune system (e.g., organ and bone marrow transplant recipients, cancerpatients and chemotherapy recipients, patients receivingimmunosuppressive drugs and HIV-infected patients) and silent HCMVinfections in otherwise healthy subjects.

Passive administration of immunoglobulins (HCMV-HIG) has showncontrasting results in clinical trials. Vaccine-derived NAbs disclosedherein, once humanized, can be used as passive immunotherapy agents tolower the transmission rate of HCMV from the mother to the fetus indocumented cases of HCMV primary infection or reactivation. In thissetting, a humanized vaccine-derived NAb can be used either alone or incombination with a human derived NAb.

In some embodiments, a method for treating or preventing CMV infectionmay include administering a therapeutically effective amount of acomposition comprising one or more vaccine-derived NAbs, such as thosedescribed herein, to a subject.

In some embodiments, a method for treating or preventing CMV infectionmay include administering a therapeutically effective amount of acomposition comprising one or more small peptides, such as thosedescribed herein, to a subject. The small peptide comprises one or moreepitopes recognized by one or more vaccine-derived NAbs.

In certain embodiments, a method for treating or preventing CMVinfection may include administering one or more doses of atherapeutically effective amount of a composition comprising one or moresmall peptides to a subject. In certain embodiments, a method fortreating or preventing CMV infection may include administering a firstdose of a therapeutically effective amount of a composition comprisingone or more small peptides to a subject and administering a second doseof a therapeutically effective amount of a composition comprising one ormore small peptides to the subject after administration of the firstdose. In certain embodiments, the second dose may be administered to thesubject about one week, about two weeks, about three weeks, about fourweeks, or about five weeks after administration of the first dose. Incertain embodiments, at least one of the doses includes administering atherapeutically effective amount of a composition comprising one or moresmall peptides, at least one of which is SEQ ID NO: 180. In certainembodiments, the first dose includes administering a therapeuticallyeffective amount of a composition comprising one or more small peptides,at least one of which is SEQ ID NO: 180. In certain embodiments, thefirst dose includes administering a therapeutically effective amount ofa composition comprising SEQ ID NO: 180 and the second dose includesadministering a therapeutically effective amount of a compositioncomprising SEQ ID NO: 179. In certain embodiments, the first and seconddose includes administering a therapeutically effective amount of acomposition comprising SEQ ID NO: 180.

“Treating” or “treatment” of a condition may refer to preventing thecondition, slowing the onset or rate of development of the condition,reducing the risk of developing the condition, preventing or delayingthe development of symptoms associated with the condition, reducing orending symptoms associated with the condition, generating a complete orpartial regression of the condition, curing the condition, or somecombination thereof. Treatment may also mean a prophylactic orpreventative treatment of a condition. Treatment using a vaccine mayresult in prevention of a disease or condition, but also may refer tothe generation of a beneficial immune response that may not necessarilyprevent the condition or treatment entirely. The treatment entailsadministering to a subject a therapeutically effective amount of avaccine-derived NAb, a composition comprising one or morevaccine-derived NAbs, a small peptide, or a composition comprising oneor more small peptides described herein.

The term “a therapeutically effective amount” or “an effective amount”as used herein refers to an amount of a substance that produces adesired effect. For example, a population of cells may be contacted oran animal may be administered with an effective amount of the pentamericcomplex to produce a desired NAb. A therapeutically effective amount ofa composition comprising an NAb or a vaccine composition comprising asmall peptide disclosed herein may be used to produce a therapeuticeffect in a subject, such as preventing or treating a target condition,alleviating symptoms associated with the condition, or producing adesired physiological effect. The precise effective amount ortherapeutically effective amount is an amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject or population of cells. This amount will varydepending upon a variety of factors, including but not limited to thecharacteristics of the substance (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication) or cells, the nature of the pharmaceutically acceptablecarrier or carriers in the formulation, and the route of administration.Further, an effective or therapeutically effective amount may varydepending on whether the substance is administered alone or incombination with another compound, drug, therapy or other therapeuticmethod or modality. One skilled in the clinical and pharmacological artswill be able to determine an effective amount or therapeuticallyeffective amount through routine experimentation, namely by monitoring acell's or subject's response to administration of a substance andadjusting the dosage accordingly. For additional guidance, seeRemington: The Science and Practice of Pharmacy, 21^(st) Edition, Univ.of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins,Philadelphia, Pa., 2005, which is hereby incorporated by reference as iffully set forth herein.

The therapeutic or vaccine compositions described herein may beadministered by any suitable route of administration. A “route ofadministration” may refer to any administration pathway known in theart, including but not limited to aerosol, enteral, nasal, ophthalmic,oral, parenteral, rectal, transdermal (e.g., topical cream or ointment,patch), or vaginal. “Parenteral” refers to a route of administrationthat is generally associated with injection, including intraorbital,infusion, intraarterial, intracapsular, intracardiac, intradermal,intralingual, intramuscular, intraperitoneal, intrapulmonary,intraspinal, intrasternal, intrathecal, intrauterine, intravenous,subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal.In one embodiment, the composition comprising a vaccine-derived NAb or asmall peptide is administered by injection.

V. Methods of Detecting CMV Infection

Screening or testing for CMV infections is very important for pregnantwomen, newborns, and immunocompromised patients. Vaccine-derived NAbsmay be used as a diagnostic tool to detect CMV antigens in humans oranimals or other biological samples or to detect CMV in cell culturesinoculated with infected samples. In particular, NAbs binding toconserved epitopes, such as 13B5, which binds a linear epitope on UL128present on all the clinical strains, are capable of detecting HCMVantigens independently of the strain.

In certain embodiments, a vaccine-derived NAb may be used to detect CMVin a biological sample obtained from a subject by contacting the samplewith the vaccine-derived NAb. The vaccine-derived NAb can be used bothas a diagnostic tool and as prognostic tool. Once the subject is testedpositive for CMV infection, the subject is administered a treatment forCMV infection and then subjected to additional tests to monitor theprogress of the treatment.

Also disclosed herein is an assay kit for diagnosing or prognosing CMVinfection in a subject including a vaccine-derived NAb and one or morereagents for performing the assay. Optionally, the kit may include aninstruction manual for performing the assay, a known CMV antigen as apositive control, and/or a negative control.

VI. Methods of Identifying Small Peptides with Potential Use as PeptideVaccines

As disclosed above, small peptides binding to linear epitopes onsubunits of the pentameric complex are particularly useful fordeveloping peptide vaccine compositions for preventing CMV infections.Vaccine-derived NAbs disclosed herein can be used to identify additionalsmall peptides comprising one or more epitopes on the pentamericcomplex.

In some embodiments, a vaccine-derived NAb is used to bind to a peptidelibrary constructed with one or more subunits of the pentameric complexin an ELISA assay. The peptides having strong binding affinity to thevaccine-derived NAbs are identified. Optionally, these peptides can befurther modified by substituting, deleting, or adding one or more aminoacid residues. The modified peptides may be tested for binding affinityto the vaccine-derived NAbs to select the peptides having improvedbinding affinity. Both the peptides without modification and themodified peptides can be used for developing peptide vaccines describedabove.

The following examples are intended to illustrate various embodiments ofthe invention. As such, the specific embodiments discussed are not to beconstrued as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope ofinvention, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entireties, as if fully setforth herein.

EXAMPLES Materials and Methods

Cells.

ARPE-19 and MRC-5 (ATCC) were maintained in Dulbecco's minimal essentialmedium (DMEM, Corning, Corning, N.Y., USA) or minimal essential medium(MEM, Corning), respectively, supplemented with 10% fetal bovine serum(FBS, HyClone, Logan, Utah, USA). HUVEC (ATCC) were grown in VascuLifeBasal Medium added with VascuLife EnGS LifeFactors (Lifeline CellTechnology, Frederick, Md., USA). BHK-21 cells (ATCC) were maintained inMEM with the addition of 10% FBS, 1 mM sodium pyruvate and 0.1 mMnon-essential amino acids (Life Technologies, Grand Island, N.Y., USA).

Isolation and Culture of CTB.

With written informed consent, term (>37 weeks gestation) placentae fromHIV-1 seronegative and Hepatitis B uninfected women (>18 years of age)were obtained immediately following elective caesarian section withoutlabor from Emory Midtown Hospital in Atlanta, Ga. Approval of the studywas granted by the Emory University Institutional Review Board (IRB).Written informed consent was obtained from donors, and samples werede-identified prior to handling by laboratory personnel. In order toisolate CTB, membrane-free villous was dissected from the placenta, aspreviously described (53-55). The tissue was thoroughly washed andmechanically dispersed in Hank's balanced salt solution (HBSS) tominimize peripheral blood contamination. Minced tissue fragments werethen subjected to three sequential enzymatic digestions in a solutioncontaining 0.25% trypsin (Mediatech Inc., Manassas, Va., USA), 0.2%DNase I (Roche Diagnostics, Mannheim, Germany), 25 mM HEPES, 2 mM CaCl₂,and 0.8 mM MgSO₄ in HBSS at 37° C. Following each digestion, theundigested tissue was removed by passage through a gauze and 100 μm cellstrainer (BD Biosciences, Franklin Lakes, N.J., USA) and washed withPBS. Supernatants from the second and third digestions were collectedand the resulting cell pellets were resuspended in 1:1 DMEM/F12supplemented with 10% FBS, 1 mM L-glutamine, and 1% pen/strep(Sigma-Aldrich, St. Louis, Mo., USA). The CTB were isolated on adiscontinuous gradient of Percoll (GE Healthcare, Uppsala, Sweden)(50%/45%/35%/30%) by centrifugation. Cells migrating to the 35%/45%Percoll interface were recovered and immunopurified by negativeselection with simultaneous treatment with anti-CD9 (to exclude EnC, FB,platelets, smooth muscle, extravillous trophoblast cells, B cells andmonocytes) and anti-CD45RA (to exclude leucocytes) antibodies andmagnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) (56, 57).The purity of the CTB population was assessed by cytokeratin-7 stainingand was on average >97%. Vimentin staining to quantify contaminationfrom mesenchymal cells was on average <3% (56-58).

Antibodies.

Cytogam (CMV-HIG, 50 mg/ml) was obtained from the manufacturer(Baxter-Healthcare Corp., Irvine, Calif., USA). The isolation of anti-gHAb AP86, anti-pp65 Ab 28-103, and anti-HCMV 1E1 Ab (p63-27) has beendescribed (59-61).

Viruses.

MVA expressing all five PC subunits (MVA-PC), single PC subunits orsubunit combinations were reconstituted from MVA-BAC as previouslydescribed (36, 62) and propagated on BHK-21 (63). For preparing MVAvirus stocks, MVA was harvested from infected BHK-21, purified by 36%sucrose density ultracentrifugation, and resuspended in 1 mM Tris-HCl(pH 9) (36, 62, 64). MVA stocks were maintained at −80° C. Purified MVAwas titrated on BHK-21 by standard procedure. HCMV strain TB40/E-GFP(TB40/E) was kindly provided by Christian Sinzger (Ulm University,Germany) (65). HCMV strain TR-GFP (TR) was a gift from Jay Nelson(Oregon Health & Sciences University, Portland, Oreg., USA). HCMVstrains Davis, Towne and AD169 were kindly provided by John Zaia(Beckman Research Institute of the City of Hope, Duarte, Calif., USA)(64). Generation of HCMV stocks was performed as previously described(36). Briefly, ARPE-19 were infected with HCMV and re-seeded until70-80% of the cells were GFP-positive. Virus particles were concentratedfrom clarified medium by ultracentrifugation (70,000×g for one hour)over 20% sucrose (w/v) in Tris-buffered saline (0.1 M Tris-CI, pH 7.4,0.1 M NaCl). Concentrated virus was resuspended in Tris-buffered salineand stored at −80° C. Virus titration was performed by adding serialdilution of the virus to ARPE-19, MRC-5, HUVEC and CTB, and byimmunostaining for immediate-early-1 protein (IE-1) after 48 hoursincubation. HCMV titer on CTB was on average three times lower than theone measured on ARPE-19.

Mice and Immunizations.

The Institutional Animal Care and Use Committee (IACUC) of the BeckmanResearch Institute of City of Hope approved protocol #98004 assigned forthis study. All study procedures were carried out in strict accordancewith the recommendations in the Guide for the Care and Use of LaboratoryAnimals and Public Health Service Policy on the Human Care and Use oflaboratory Animals. Methods of euthanasia followed “Report of the AVMAPanel on Euthanasia”(http://www.avma.org/kb/policies/documents/euthanasia.pdf). BALB/cJ mice(Jackson Laboratory, Bar Harbor, Me., USA) were vaccinated with MVA-PCas previously described (36) and boosted four days before the spleen wasremoved for hybridoma production.

Hybridoma Derivation.

Hybridomas were derived by conventional procedure (66). Briefly, myelomapartner cells (P3X63Ag8.653, ATCC) were maintained in RPMI-1640(Corning) supplemented with 10% FBS. Splenocytes and myeloma cells werecounted, and fusion was performed at a 1:5 ratio by adding 1 ml PEG 1500(Sigma-Aldrich). After centrifugation, fused cells were resuspended inRPMI-1640 supplemented with 15% FBS, 10% UltraCruz Hybridoma CloningSupplement (HCS, Santa Cruz Biotechnology, Santa Cruz, Calif., USA), HATmedia supplement (Sigma-Aldrich) at a concentration of 5×10⁵splenocytes/ml. Cells were seeded in 96 well plates and incubated in a5% CO₂, 37° C. incubator. Selected hybridoma clones were grown inRPMI-1640 supplemented with 15% FBS and 10% HCS. Each clone underwent 2rounds of single cell subcloning to ensure the clonality of the antibody(66). Collected hybridoma supernatant was added with 20 mM sodiumphosphate buffer (pH 7.0) and NAbs were purified using a HiTrap ProteinG HP 5 ml column (GE Healthcare) according to the manufacturer'sinstructions. Ab concentration was verified with Bradford-Coomassiebrilliant blue dye method using a bovine gamma globulin standard (ThermoScientific/Pierce, Rockford, Ill., USA).

Neutralization Assays.

Cells were seeded at 1.5×10⁴ cells/well (ARPE-19, HUVEC and MRC-5) or1.5×10⁵ cells/well (CTB) in a clear bottom 96-well plate (Corning).Around 24 hours later the medium in every plate was replaced with 50 μlper well of fresh growth medium. Naturalization assays were performed aspreviously described (36). Briefly, serial two-fold dilutions of thepurified NAbs were prepared in complete growth medium in a final volumeof 75 μl. NAb dilutions were mixed with 75 μl of complete growth mediumcontaining approximately 9000 PFU of HCMV TB40/E or TR and incubated for2 h at 37° C. The mixture was transferred to the cells (50 μl each,duplicate wells). After 48 hours, cells were fixed and IE-1immunostaining performed as previously described (36). NAb concentrationinhibiting 50% of the virus infectivity (IC50) was calculated aspreviously described (36).

Antibody Purification.

Hybridoma clones of interest were grown in RPMI-1640 supplemented with15% FBS and 10% HCS. Each clone underwent 2 rounds of single cellcloning to ensure the monoclonality of the antibody. After cloning, thehybridomas were grown in RPMI-1640 supplemented with 15% FBS.Periodically, half of the medium was collected and replaced with freshone. When the collected hybridoma supernatant was about 500 ml, 200 mlof 20 mM sodium phosphate buffer (pH 7.0) were added and the antibodywas purified using a HiTrap Protein G HP 5 ml column (GE Healthcare,Piscataway, N.J., USA) according to the manufacturer's instructions.Concentration of the antibody was verified with Bradford-Coomassiebrilliant blue dye method using a bovine gamma globulin standard (bothThermo Scientific/Pierce, Rockford, Ill., USA).

NAb Binding Specificity.

NAb subunit specificity was evaluated by staining BHK-21 cells infectedwith different MVA recombinants. One or more vectors were used toco-infect BHK-21 at an MOI of 5. The combinations analyzed were: UL128,UL130, UL131A, UL128/130, UL128/131A, UL130/131, UL128/130/131A, gH,gH/gL, gH/gL/UL128, gH/gL/UL130, gH/gL/UL131A, gH/gL/UL128/130,gH/gL/UL128/131A, gH/gL/UL130/131A and gH/gL/UL128/130/131A. Four hourspost infection cells were fixed and permeabilized using Cytofix/Cytopermsolution (BD Biosciences). NAbs (1 mg/ml) were diluted 1:500 inperm/wash buffer (BD) and added to the cells for 1 hour at 4° C. Afterwashing with perm/wash buffer, Alexa Fluor 647 Goat Anti-Mouse IgG (LifeTechnologies) was added at a dilution of 1:2,000. Cells were washedagain and resuspended in PBS/0.1% BSA. Fifteen thousand events werecollected using the Gallios Flow Cytometer (Beckman Coulter, Miami,Fla., USA) and analyzed with FlowJo Software (Tree Star, Ashland, Oreg.,USA). Uninfected cells and cells infected with MVA-Venus were used ascontrols. GFP expression was analyzed for confirming MVA infection sinceall the constructs contain a GFP expression cassette (36, 62).

Cell-to-Cell Spread Inhibition Assay.

NAbs ability to inhibit cell-to-cell spread and/or syncytia formationwas evaluated on EpC using TB40/E and TR. ARPE-19 cells were seeded on ablack 96-well plate (Costar) and infected 24 hours later with HCMVTB40/E or TR (MOI of 1 as titrated on ARPE-19). After incubation for 24hours, cells were extensively washed with PBS and two-fold serialdilutions of each NAb were added to the wells in a total volume of 200μl. After 8 days incubation, the plates were imaged with a ZeissAxiovert fluorescence microscope (Carl Zeiss, Jena, Germany) andcellular GFP was quantified using ImagePro Premier Software (MediaCybernetics, Silver Spring, Md., USA). The percent of spread inhibition(IC) for each dilution was calculated as: IC=[1−(fluorescence ininfected wells incubated with NAb)/(fluorescence in infected wellswithout NAb)]×100. 50% cell-to-cell spread inhibition (IC50) wascalculated by determining the linear slope of the graph plotting ICversus NAb dilution by using the next higher and lower IC values thatwere closest to 50%.

Antibody Binding Affinity.

Antibody binding affinity was determined as described (67). Briefly, 10mg of purified NAbs were biotinylated using EZ-Link NHS-PEG4-BiotinBiotinylation Kit (Thermo Scientific/Pierce) following manufacturer'sinstructions. BHK-21 cells were infected with MVA-PC at an MOI of 5.After an incubation of 4 hours at 37° C., the cells were dispensed at aconcentration of 1×10⁵ cells/well in a 96-well V-bottom plate, followedby 2 hours incubation at 4° C. with two-fold serial dilutions of thebiotinylated NAb in PBS/0.1% BSA. Dilutions ranged from 500 μg/ml to 0.1ng/ml. Cells were washed twice with PBS/0.1% BSA and incubated for 1hour at 4° C. in the presence of streptavidin-DyLight 650 (ThermoScientific) diluted 1:500 in PBS/0.1% BSA. After washing twice, cellswere fixed with 4% paraformaldehyde. Fifteen thousand events wereacquired with the Gallios Flow Cytometer and analyzed with FlowJoSoftware. The equilibrium binding constant (Kd) was derived by plottingfluorescence as a function of the logarithm of NAb concentration toobtain a sigmoidal curve analyzed with the 4 Parameter Logistic (4PL)nonlinear regression model (GraphPad Prism 6 Software, San Diego,Calif., USA).

Competition Assay.

NAb competition was evaluated as follows. BHK-21 cells were infectedwith MVA-PC at an MOI of 5 and 4 hours later treated withCytofix/Cytoperm. Around 1×10⁵ cells were incubated for 2 hours with 20to 100-fold excess unlabeled competitor NAb (from 100 to 200 μg/ml).After washing with perm/wash buffer cells were incubated for 2 hours inthe presence of 1 to 5 μg/ml biotinilated NAbs. For every NAb, cells inwhich the unlabelled competitor was not added to the biotinilated NAbswere used to determine maximum binding. Cells were washed once withperm/wash buffer and incubated for 1 hour with streptavidin-DyLight 650diluted 1:500. After a final washing step, cells were resuspended inPBS/0.1% BSA, acquired with Gallios Flow Cytometer and analyzed withFlowJo Software. For every antibody pair, the percentage of inhibitionwas calculated as: 100−[(% fluorescent cells with competitor NAb/%fluorescent cells without competitor NAb)×100]. The complete preventionof binding of a biotinilated NAb by its unlabelled counterpart was usedas a validation of the assay.

NAb Variable Heavy and Light Chain Sequence Characterization.

Total RNA was extracted from hybridomas using the SV total RNA isolationsystem (Promega, Madison, Wis., USA). cDNA was generated by randomhexamers (Qiagen GmbH, Hilden, Germany) and Superscript III reversetranscriptase (Life Technologies) following the manufacturer'sinstruction. The kappa variable genes were characterized by a 5′RACE PCRin which the cDNA was tailed with poly dGTP by terminal transferase (NewEngland BioLabs, Ipswich, Mass., USA). A 3′ reverse gene-specific primerlocated in the kappa constant region near the variable region(TGGATGGTGGGAAGATGGATACAGT) (SEQ ID NO: 183) was adopted together withpoly dCTP to amplify the kappa variable genes. For the gamma variablegenes, a protocol from Fields et al. (68) was followed. V_(H) and V_(L)genes were amplified by Phusion high-fidelity DNA polymerase (ThermoScientific) and cloned into pCR4Blunt-TOPO vector (Life Technologies)following the manufacturer's instruction. Three clones derived from eachV_(H)/V_(L) genes were sequenced.

Immunoblot.

Immunoblot to determine NAbs binding to denaturated gH was performedusing lysates from cells infected with a gH-expressing adenoviral vector(Ad-gH) as previously described (36). Anti-gH Ab AP86 (59), 18F10, 21E9,62-11, 62-100 and 2-80 were employed at a dilution of 10 μg/ml.Anti-MEK1/2 (Cell Signaling Technology, Danvers, Mass., USA) was diluted1:1000. Immunoblot to evaluate 18F10 and AP86 binding to lysates fromcells infected with different HCMV strains was performed as describedabove with the difference that lysates consisted in 2.5×10⁵ MRC-5infected for 4 days with an MOI of 1 of HCMV strain Davis, Towne, AD169,TB40/E or TR. Anti-pp65 was used to show HCMV infection in all thesamples independently from the strain used.

Example 1. Isolation of Durable HCMV Specific NAbs from MVA-gH/gL-PCVaccinated Mice

It was previously demonstrated that mice vaccinated three times by fourweek interval with MVA-gH/gL-PC develop high titer EpC specific NAbresponses that remained stable over at least one year (36). Remarkably,only two vaccinations with MVA-gH/gL-PC appeared to be sufficient toinduce maximum high titer EpC NAb titers. In order to characterize indetail the specific activity of the antibody response induced byMVA-gH/gL-PC, a panel of monoclonal NAbs was isolated after induction ofanamnestic responses by a fourth dose of vaccination forty-eight toseventy-two weeks after initial immunization. NAb titers after thefourth vaccination could be boosted to levels that were observed afterthe second or third boost with MVA-gH/gL-PC at week four and eight (FIG.1), suggesting that vector immunity had only little influence on theability of MVA-gH/gL-PC to boost NAbs to gH/gL-PC. Splenocytes wereimmortalized by conventional hybridoma technology (66) and hybridomasupernatants were screened directly by ARPE-19 EpC basedmicroneutralization using HCMV strain TB40/E for infection. Ten NAbs(1B2, 54E11, 21F6, 12E2, 1365, 18F10, 21E9, 62-11, 62-100 and 2-80) thatblocked HCMV infection of ARPE-19 were identified. NAbs were subcloned,purified by affinity chromatography, and characterized as describedbelow.

Example 2. MVA-PC Vaccine-Derived NAbs Recognize Epitopes of the PC andgH

PC-specific NAbs isolated from chronically infected HCMV⁺ individualspredominantly recognize conformational antigenic sites formed byUL130/131A and UL128/130/131A (32, 33). Only one human NAb has beenpublished that recognizes an epitope within the UL128 subunit (32). Apanel of NAbs from mice immunized with the MVA-PC vaccine was isolatedby conventional hybridoma technology combined with screening forneutralization against TB40/E on ARPE-19 EpC. In order to determine theantigen specificity of the vaccine-derived NAbs, intracellular flowcytometry (FC) staining of permeabilized BHK-21 cells infected with MVAexpressing single subunits or combinations of two or more subunits ofthe PC was evaluated. Consistent with human NAbs, four vaccine-derivedPC-specific NAbs that recognized quaternary epitopes formed byUL130/UL131A or UL128/130/131A, and one NAb (13B5) with UL128specificity were identified (FIG. 2 and Table 2). Staining with 1B2 and12E2 NAbs was observed with UL128/130/131A or all five PC subunits.Expression of single subunits or any PC subunit combination with onlyone or two of the UL128/130/131A subunits did not result in binding of1B2 and 12E2. In contrast, NAbs 54E11 and 21F6 showed binding withUL130/131A, the three UL128/130/131A subunits, or all five PC subunits.Single subunits or PC subunit combinations lacking UL130/131A failed toenable binding of 54E11 and 21F6. NAb 13B5 showed binding with UL128alone or combined with other PC subunits, whereas binding of 13B5 wasnot observed in the absence of UL128. Based on the vaccine's ability toelicit both EpC/EnC and FB specific NAbs (36), NAbs with gH specificitywere also identified. Staining by these NAbs was confirmed with gHalone, in combination with gL, or together with all other four PCsubunits. Binding of these antibodies was not observed when gH wasmissing (FIG. 2 and Table 2). Hence, MVA-PC elicits PC- and gH-specificNAbs that have antigen recognition patterns similar to human NAbsisolated from chronically infected HCMV⁺ individuals.

TABLE 2 NAb subunits recognition UL128 UL128 UL128 UL130 UL130 UL128UL130 UL131 UL130 UL131 UL131 UL131 gH gH/gL 1B2 − − − − − − + − − 54E11− − − − − + + − − 21F6 − − − − − + + − − 12E2 − − − − − − + − − 13B5 + −− + + − + − − 18F10 − − − − − − − + + 21E9 − − − − − − − + + 62-11 − − −− − − − + + 62-100 − − − − − − − + + 2-80 − − − − − − − + + 4A3 − − − −− − + − − 6G2 − − − − − − − + + 10G6 − − − − − − − + + 13B1 − − − − − −− + + 25H10 − − − − − − − + + gH/gL gH/gL gH/gL gH/gL UL128 gH/gL gH/gLgH/gL UL128 UL128 UL130 UL130 UL128 UL130 UL131 UL130 UL131 UL131 UL1311B2 − − − − − − + 54E11 − − − − − + + 21F6 − − − − − + + 12E2 − − − − −− + 13B5 + − − + + − + 18F10 + + + + + + + 21E9 + + + + + + +62-11 + + + + + + + 62-100 + + + + + + + 2-80 + + + + + + + 4A3 − − − −− − + 6G2 + + + + + + + 10G6 + + + + + + + 13B1 + + + + + + +25H10 + + + + + + +

Example 3. MVA-PC-Infected Cells Present PC- and gH-SpecificNeutralizing Epitopes at the Cell Surface

Although it was reported that the five PC subunits expressed from MVA-PCassemble with each other intracellularly, it was unclear whether thecomplexes were transported to the cell surface and presented PC-specificneutralizing epitopes. The vaccine-derived NAbs for cell surface FCstaining of live non-permeabilized BHK-21 cells infected with MVA-PC wascompared to MVA vaccine vectors expressing single subunits or differentsubunit subset combinations of the PC. When compared to intracellularstaining (FIG. 2A), different cell surface recognition patterns with thevaccine-derived PC-specific NAbs were observed (FIG. 2B). Intensive cellsurface staining by the PC-specific NAbs was confirmed with all five PCsubunits (MVA-PC), whereas no or only minimal cell surface staining byNAbs was observed with single subunits or subunit subsets of the complex(FIG. 2B). As confirmed for intracellular staining (FIG. 2A), intensecell surface staining by all gH-specific NAbs with gH alone, togetherwith gL, or combined with all other four PC subunits was observed. Incontrast to intracellular staining, cell surface staining by the anti-gHNAbs was more intense with all five PC subunits when compared to gHalone or only gH/gL. In addition, compared to gH single expression,stronger binding of the anti-gH NAbs was observed with gH/gL (FIG. 2B).These results demonstrate that the five PC subunits expressed fromMVA-PC efficiently assemble with one another and present conformationalneutralizing epitopes of the UL128/130/131A subunits and gH at the cellsurface.

Example 4. Vaccine-Derived PC-Specific NAbs are More Potent thangH-Specific NAb in Neutralizing HCMV

In order to determine whether the vaccine-derived NAbs confer similarpotency than previously described human NAbs (32) to prevent host cellentry, the inhibitory antibody concentration (IC50) that blocked 50%HCMV infection of ARPE-19 EpC, HUVEC EnC, or MRC-5 FB was evaluatedusing a standard microneutralization assay. Neutralization against HCMVstrains TB40/E and TR was tested to evaluate whether sequence variationin the gH component influences the potency of the NAbs to neutralizeHCMV (36, 69). Neutralization potency of HCMV hyperimmune globulin(CMV-HIG) was evaluated as a reference. All PC-specific NAbs blockedTB40/E or TR infection of ARPE-19 cells and HUVEC with potency thatsignificantly exceeded (on average over 200-fold) that of anti-gH NAbsor CMV-HIG (FIG. 3 and Table 3). In contrast, most of the gH-specificNAbs inhibited HCMV infection of all investigated cell types withcomparable potency, albeit with much lower potency than the PC-specificNAbs blocked ARPE-19/HUVEC entry. Neutralization potency of the anti-gHNAbs was similar to that determined for CMV-HIG. Neutralization withCMV-HIG on MRC-5 FB at the highest investigated concentration (25 μg/ml)was not observed, which is consistent with observations obtained byothers (37, 70). One gH-specific NAb (18F10) demonstrated inhibitionpotency comparable to the other anti-gH NAbs when measured on ARPE-19cells and HUVEC, but it did not show ability to block HCMV infection ofFB. The IC50 values of the vaccine-induced PC and gH-specific NAbsisolated were similar to published values determined for NAbs isolatedfrom HCMV⁺ individuals (Table 3). In contrast to the other anti-gH NAbs,two of the gH-specific NAbs (62-11, 62-100) blocked TR infection lesspotently than infection of TB40/E (FIG. 3), suggesting that anti-gH NAbs62-11 and 62-100 may target epitopes that are antigenically distinct inTB40/E and TR. These results show that the vaccine-derived PC- andgH-specific NAbs have neutralization potency comparable to that observedfor NAbs from chronically HCMV infected individuals.

TABLE 3 Potency of NAb in preventing HCMV TB40/E and TR infection ofdifferent cell types and epithelial cell-to-cell spread and theirbinding affinity to the PC TB40/E TR IC50 IC50 IC50 IC50 EC50 (ng/ml)(μg/ml) (ng/ml) (μg/ml) (M) Specificity/ APRE- APRE-19 APRE- APRE-19Binding NAb 19 HUVEC MRC-5 CTB Spread 19 HUVEC MRC-5 Spread affinity UL1B2 0.6 0.5 >25000 12.6 1 0.3 0.7 >25000 1.4 3.2E−10 54E11 3.44.6 >25000 24.9 17 3.4 5.9 >25000 18 5.1E−10 21F6 2.6 4.0 >25000 30.7 183.5 5.9 >25000 19 6.2E−10 12E2 3.7 6.0 >25000 72.1 15 3.1 4.2 >25000 171.1E−09 13B5 15 12 >25000 99.8 22 19.9 7.7 >25000 21 6.8E−10 4A3 80.1546.42 >25000 44.6 10.15 >25000 gH 18F10 763 1490 >25000 8800 95 885675 >25000 78 6.5E−09 21E9 1409 2374 5780 42362 157 926 2916 4141 2621.8E−08 62-11 185 102 182 2760 87 641 830 3346 395 1.7E−09 62-100 270156 293 3623 103 2126 1031 1983 337 3.3E−09 2-80 1433 14051597 >50000 >400 2125 2540 1717 398 2.8E−09 6G2 689 542 1255 6800 10G6326 481 706 6315 13B1 3022 6993 25H10 188 602 1076 6235 CMV-HIG 10861124 >25000 5478 271 991 2413 >25000 299 ND gH AP86 1005 ND >25000 ND NDND ND ND ND ND In ARPE-19, HUVEC and MRC-5 neutralization assays themaximum Ab concentration tested was 25 μg/ml. In CTB neutralizationassay the maximum Ab concentration tested was 50 μg/ml. In cell-to-cellspread assay the maximum Ab concentration was 400 μg/ml. ND = not done.

Example 5. Vaccine-Derived NAbs Recognize Conformational Epitopes

Most of the potent NAbs of HCMV+ individuals that block HCMV infectionof EpCs target conformational epitopes constituted by two or moresubunits of gH/gL-PC, but mainly epitopes formed by the UL128-UL131Asubunits. In order to determine similar gH/gL-PC subunit specificity forthe vaccine-derived NAbs, intracellular antibody recognition of singlesubunits, different combination of two or more subunits, or all fivesubunits of gH/gL-PC expressed from MVA was performed. Baby hamsterkidney (BHK) cells that allow efficient MVA replication were infectedwith the different MVA constructs, fixed and permeabilized, and analyzedby flow cytometry (FC) for staining by the isolated NAbs. All potentEpC/EnC neutralizers recognized antigenic sites that requiredco-expression of more than one subunit of UL128-UL131A, except oneantibody (13B5) that showed specificity for UL128 (FIG. 2 and Table 2).NAbs 1B2 and 12E2 showed minimal antigen recognition forUL128/UL130/UL131A. These antibodies stained BHK cells infected withMVA-gH/gL-PC or MVA co-expressing UL128, UL130, and UL131A, thoughsingle subunits or any other subunit combination of gH/gL-PC wereundetected (FIGS. 1 and 4). EpC/EnC neutralizer 54E11 and 21F6demonstrated minimal antigen specificity for UL130/UL131A. Theseantibodies recognized co-expression of all five gH/gL-PC subunits,UL128/UL130/UL131A, or UL130/UL131A, whereas cells infected with MVAexpressing single subunits or other gH/gL-PC subunit combinationsremained unstained (FIG. 2 and Table 2). Antibody 13B5 with specificityfor UL128 recognized UL128 single expression or UL128 expressiontogether with any other combination of gH/gLPC subunits, but did notstain cells infected with MVA expressing only UL130, UL131A, or gH/gL(FIG. 2 and Table 2). All the NAbs that blocked EpCs/EnCs and FBs HCMVinfection (21E9, 62-11, 62-100, 2-80, 1361, 6G2, 10G6 and 25H10) showedspecificity for gH, gH/gL and all five gH/gL-PC subunits expressed fromMVA, whereas no staining was observed when any combination of the UL128to UL131A was expressed without gH (FIG. 2 and Table 2). These antigenrecognition patterns are strikingly similar to that of natural NAbs togH/gL-PC. Finally, despite unable to neutralize HCMV entry of FBs, NAb18F10 showed a minimal binding specificity for gH, and also bound whencells were infected with MVA-gH/gL-PC. This result shows for the firsttime the existence of an NAb specific for gH but neutralizing only entryinto EpCs/EnCs.

Example 6. PC-Specific NAbs Limit HCMV Spread in EpC More Potently thanAnti-gH NAbs

HCMV replication is highly cell-associated and the virus predominantlyspreads from cell to cell (71, 72). Inhibition of HCMV cell-to-cellspread and/or syncytia formation besides neutralization of cell-freeHCMV entry may be an important antibody function to preventdissemination. To test whether the MVA-PC vaccine-derived NAbs can blockspread of HCMV, the potency to inhibit HCMV TB40/E or TR cell-to-cellspread and/or syncytia formation in ARPE-19 EpC was evaluated. As shownin FIG. 3 for the neutralization potency, significant differences in thepotency of the PC- and gH-specific NAbs to prevent HCMV cell-to-cellspread were found (FIG. 4 and Table 3). All NAbs specific for PCsubunits blocked TB40/E and TR spread in ARPE-19 cells with potency thatsignificantly exceeded those of the anti-gH NAbs or CMV-HIG. Incontrast, the anti-gH NAbs demonstrated only very low spread inhibitionpotency, or were even unable to prevent HCMV spread in ARPE-19 cells ata cut-off concentration of 400 pg/ml. Most of the anti-gH NAbsdemonstrated spread inhibition potency that was comparable to or onlyslightly higher than those of CMV-HIG. The ability of individual NAbs toblock viral spread in ARPE-19 EpC was similar to their property toneutralize HCMV infection of these cells, with the variation that higherantibody amounts (1000-fold) were required to interfere with HCMVcell-to-cell spread than with HCMV entry (Table 3). Consistent with theneutralization by the anti-gH NAbs, less potent spread inhibition ofHCMV TR than of TB40/E by two of the anti-gH NAbs (62-11 and 62-100) wasobserved, suggesting that sequence variation in the gH proteininfluences the ability of the NAb to prevent HCMV spread. FIG. 5 showsthe effect of potent PC-specific NAb 1B2 and anti-gH NAbs 21E9 and 62-11compared to untreated controls to interfere with TB40/E and TR spreadand/or syncytia formation in ARPE-19 cells. These results indicate thatPC-specific NAbs are more potent than anti-gH NAbs to prevent HCMVspread in EpC.

Example 7. Neutralization Potency of Vaccine-Induced NAbs May Correlatewith Antibody Affinity

It has been shown that a reduced risk for HCMV transmission to the fetusis associated with high affinity, highly neutralizing antibody responses(42, 73). Consequently, the affinity of the vaccine-induced NAbs to bindcell surface PC on MVA-PC infected BHK-21 cells was evaluated andwhether the antibody affinity correlates with potency to neutralize HCMVwas investigated. FIG. 6A shows a positive correlation between antibodyaffinity and neutralization potency taking into account all isolatedNAbs (r=0.743, p=0.014). Consistent with the neutralization potency,higher binding affinity was observed with the PC-specific NAbs than withthe anti-gH NAbs (FIG. 6A and Table 3). In addition, the highestaffinity was observed with 1B2, which is the most potent NAb to blockEpC/EnC entry identified (Table 3). However, despite a significantdifference in neutralization potency between PC- and gH-specific NAbs(p=0.0167, FIG. 6C), the difference in binding affinity of thePC-specific NAbs and anti-gH NAbs was not significant (p>0.05, FIG. 6B).The EC50 values determined for the vaccine-derived NAbs were in therange of published values for HCMV NAbs observed by others (34). Thesefindings provide evidence that the neutralization potency ofvaccine-derived PC and gH-specific NAbs correlates with their ability tobind the PC.

Example 8. Vaccine-Derived PC- and gH-Specific NAbs Recognize DifferentAntigenic Target Sites

It has been reported that human NAbs recognizing the PC target at leastseven distinct antigenic sites (32, 33). In order to determine whetherthe vaccine-induced NAbs bind overlapping or non-overlapping targetsites of the PC, the ability to cross-compete for binding to PCexpressed in MVA-PC infected BHK-21 cells was evaluated. As shown inTable 4, binding competition was observed between the two PC-specificNAbs 1B2 and 12E2, indicating that 1B2 and 12E2 recognize overlappingtarget sites formed by UL128/130/131A. The same result was obtained withthe PC-specific NAbs 54E11 and 21F6, demonstrating that these NAbstarget similar binding sites constituted by UL130/131A.UL128/130/131A-specific NAbs and UL130/131A-specific NAbs did notcompete for binding with each other or with the anti-UL128 NAb 13B5.Binding competitions between the anti-gH NAbs 62-11 and 62-100 andbetween anti-gH NAbs 21E9 and 2-80 were observed. Hence, 62-11 and62-100 or 21E9 and 2-80 target similar antigenic sites on gH. Inaddition, 62-11 and 62-100 demonstrated ability to partially compete forbinding with 21E9 and 2-80, suggesting that 62-11 and 62-100 sharepartially overlapping binding sites on gH with 21E9 and 2-80. Incontrast to all other isolated gH NAbs, NAb 18F10 was not able tocompete with any of the gH-specific NAbs. Overall three antigenic siteson the UL128/130/131A subunits and three antigenic sites on gH wereidentified (Table 4). The VH and VL genes from each NAb was sequenced asfollows: mRNA was isolated from hybridoma cells and reverse transcribedinto cDNA. Next, VH and VL regions were amplified by PCR, gel purified,ligated into a standard cloning vector and clones selected from LBplates. Multiple clones were selected for sequencing and the finalsequence was confirmed by at least three identical sequencing results.Complementarity determining regions (CDRs) sequencing revealed thatEpC/EnC NAbs 54E11 and 21F6, both binding to the same antigenic site onUL130/131A, are encoded by the same sequence (Table 5).

Although some of the NAbs competed for the same antigenic site, uniquevariable heavy (V_(H)) and light (V_(L)) chain sequences for most of theNAbs were determined (Table 5). Despite sharing the same antigenicbinding site on UL128/130/131A, NAbs 1B2 and 12E2 have completelydifferent CDR sequences. Of the gH NAbs competing for the same antigenicsite, 62-11 and 62-100 share similar, but not identical, CDR sequences.Identical V_(H) and V_(L) sequences were only observed for the twoUL130/131A-specific NAbs 21F6 and 54E11. It was confirmed that 54E11 and21F6 have different isotypes (Table 5), suggesting that these NAbs werederived from the same centroblast B cell after class switchrecombination (74). A very limited number of point mutations in V_(H)and V_(L) sequences of the NAbs when compared to germ line sequenceswere identified (Table 5), suggesting that, at least in immunized mice,potent HCMV NAbs are already encoded by the germline with very lowinfluence of affinity maturation. This data indicate thatvaccine-derived NAbs recognize predominantly distinct antigenic targetsites on the UL128/130/131A subunits or gH.

TABLE 4 NAb competition for binding to the PC Unlabelled BindingInhibition (%) Biotinilated Abs Antigenic Ab 1B2 54E11 21F6 12E2 13B518F10 21E9 62-11 62-100 2-80 AP86 site* UL 1B2 100 0 0 100 0 0 ND ND NDND ND 1UL NAb 54E11 0 100 100 0 0 0 ND ND ND ND ND 2UL 21F6 0 100 100 00 0 ND ND ND ND ND 2UL 12E2 100 0 0 100 0 0 ND ND ND ND ND 1UL 13B5 0 00 0 100 0 ND ND ND ND ND 3UL gH 18F10 0 0 0 0 0 100 0 0 0 0 100 1gH NAb21E9 ND ND ND ND ND 0 100 32 28 100 0 2gH 62-11 ND ND ND ND ND 0 46 100100 54 0 3gH 62-100 ND ND ND ND ND 0 51 100 100 57 0 3gH 2-80 ND ND NDND ND 0 100 43 40 100 0 2gH AP86 ND ND ND ND ND 100 0 0 0 0 100 1gH ND =not done; *antigenic site numbers are arbitrarily assigned based oncross-competition.

TABLE 5 NAb binding affinity and sequence analysis Isotype (class andsubclass, VL % VH %* light chain VL/VH CDR3 sequences Mut % Mut %Antibody Specificity type) VL CDR3 VH CDR3 aa aa mut aa aa mut 1B2UL128/130/131 IgG1, k QQSNRWPWT ARGWLLPVFAY 107 5 4.7 118 4 3.4 54E11UL130/131 IgG1, k QQYSKLPYT AREHYYGINPLLGC 107 1 0.9 121 1 0.8 21F6UL130/131 IgG2a, k QQYSKLPYT AREHYYGINPLLGC 107 1 0.9 121 1 0.8 12E2UL128/130/131 IgG2a, k QHSRELPWT VRPKRDFQYLYAMDY 111 2 1.8 122 6 4.913B5 UL128 IgG2a, k QNGHTFPPT VRSLYDYDEGYYFDS 107 4 3.7 123 16 13.018F10 gH IgG1, k SQSTHVPYT ARTGYFDV 112 6 5.4 116 9 7.8 21E9 gH IgG2a, kQQDYSSPWT ARKGYYGSSGYFDY 107 1 0.9 121 2 1.7 62-11 gH IgG1, k QQYSKLPYTSNGYSSFAY 107 2 1.9 116 7 6.0 62-100 gH IgG1, k QQSNSWPLT SNGYSSFAY 1072 1.9 116 7 6.0 2-80 gH IgG2a, k QQSNEDPLT ARRGDGLYSMDY 111 2 1.8 119 21.7 *Mutations brought in by the VH primers (68) were excluded.

Example 9. NAb 18F10 Binds an Immunodominant Linear Epitope on gH

Since all identified gH-specific NAbs showed binding to gH byintracellular and cell surface staining even in the absence of gL (FIG.2 and Table 2), whether the anti-gH NAbs bind linear or conformationalepitopes on the gH protein was investigated. Therefore, the NAb wastested to recognize gH expressed from adenoviral vectors (AdV) usingimmunoblot analysis under denaturing conditions. As a control, thewell-characterized anti-gH antibody AP86 was used, which is known tobind the linear immunodominant neutralizing epitope of gH(34-LDPHAFHLLL-43) (SEQ ID NO: 184) (59). Compared to AP86, only anti-gHNAb 18F10 efficiently recognized denatured gH, while all othergH-specific NAbs demonstrated only minimal ability to react with thelinear form of gH (FIG. 7A). Based on this observation, it is possiblethat 18F10 like AP86 binds the linear immunodominant epitope of gH. Toobtain further evidence for the similar antigen recognition propertiesof AP86 and 18F10, the neutralization potency of 18F10 and AP86 to blockTB40/E infection of MRC-5 FB or ARPE-19 EpC was determined. Differentfrom previous reports (59), although comparable to what was observed forvaccine-derived anti-gH NAb 18F10, AP86 was unable to neutralize entryof TB40/E into MRC-5 FB (Table 3). However, AP86 had comparable potencyto 18F10 to prevent entry of TB40/E into ARPE-19 cells (IC50 1 μg/ml.Table 3). Since the AP86 epitope is present in most HCMV strains but notin Towne due to a gap and a point mutation (34-LD*KAFHLLL-43) (SEQ IDNO: 185) (59), 18F10 and AP86 for recognition of gH in Towne, T R,Davis, AD169 and TB40/E infected MRC-5 cells were evaluated viaimmunoblot. Both 18F10 and AP86 bound to gH from TR, Davis, AD169 andTB40, but they did not bind to Towne gH (FIG. 7B). Finally, crosscompetition of AP86 with 18F10 was evaluated and, as a control, alsowith all other gH-specific NAbs to recognize the PC in MVA-PC infectedBHK-21 cells. Only 18F10 competed with AP86 for binding to the PC (Table4). An ELISA assay was performed with the TB40 specific peptide, L10L,(i.e., (LDPHAFHLLL) (SEQ ID NO: 184)) that binds AP86 and with the Townespecific peptide, L9L, (i.e., (LD*KAFHLLL) (SEQ ID NO: 185)) that doesnot bind AP85. Results show that despite binding to the same antigenicsite, 18F10 and AP86 did not bind the same epitope (see FIG. 9).Therefore 18F10 binds to a novel epitope that was not previouslydescribed. These data indicate that binding of the vaccine-derivedanti-gH NAb 18F10 overlaps with the linear immunodominant epitope ofHCMV gH and, hence, provides further evidence that the NAb responsesinduced by MVA-PC are similar to those induced during natural HCMVinfection.

Example 10. PC-Specific NAbs are More Potent than Anti-gH NAbs to BlockHCMV Infection of Placental CTB

CTB are thought to be the key placental cells HCMV utilizes to cross thefetal-maternal interface (40, 41, 46). In order to determine whether thevaccine-derived NAbs can block HCMV infection of CTB, a standardmicroneutralization assay was used to evaluate the neutralizationpotency against TB40/E using freshly prepared primary CTB from termplacentae. As shown in FIG. 8A, the prepared cell populations werealmost exclusively positive for cytokeratin 7 and negative for vimentin,showing that almost all cells were primary CTB, while only minorproportions accounted for mesenchymal cells (57, 58, 75). Consistentwith the potency to neutralize HCMV entry and cell-to-cell spread, theneutralization potency of all PC NAbs measured on CTB was significantlyhigher than that of the NAbs targeting gH or CMV-HIG (FIG. 8B and Table3). Compared to all NAbs isolated, the highest potency to block CTBinfection was demonstrated with PC-specific NAb 1B2, which had also themost potent ability to inhibit HCMV entry into EpC and spreading inthese cells. In contrast, anti-gH NAbs showed lower level neutralizationpotency against TB40/E on CTB that was only comparable to that ofCMV-HIG. Some anti-gH NAbs were even unable to prevent TB40/E infectionof CTB at the highest investigated antibody concentration (50 μg/ml).These data demonstrated that NAbs specific for the UL128/130/131Asubunits of the PC confer higher protection against HCMV infection ofprimary CTB from term placentae than NAbs targeting gH or antibodypreparations from HCMV⁺ individuals.

Example 11. NAb 13B5 Binding Site

The NAb 1385 Binding Site within UL128 is at Minimum 13 Amino Acids inLength.

Linear B cell epitopes vary greatly in length and range from 5-22 aminoacids (1, 2) with an average of 15 (3). In order to define the aminoacid sequence within UL128 that constitute the 13B5 epitope, binding of13B5 to N-terminal and C-terminal truncated sequences of peptide 40 ofthe UL128 peptide library was evaluated via ELISA. Peptide 40 iscomposed of amino acids 157-KRLDVCRAKMGYMLQ-171 of the UL128 protein,and demonstrated strongest binding to the 13B5 antibody and hence waslikely to contain the minimal 13B5 binding sequence. Removal of theN-terminal amino acid K and sequential removal the four following aminoacids (RLDV) from the C-terminus of peptide 40 resulted in dramaticallyreduced 13B5 binding. Complete loss of 13B5 binding was observed byremoving six or more N-terminal amino acids from peptide 40 (FIG. 10A).Truncation of the two C-terminal amino acids LQ from peptide 40 did notshow a reduction in 13B5 binding, though slightly decreased 13B5 bindingwas observed by additionally removing the M from the peptide 40C-terminus (FIG. 10B). 13B5 binding was substantially decreased byremoving the four C-terminal amino acids YMLQ from peptide 40, and wascompletely lost when five or more C-terminal amino acids of peptide 40were removed (FIG. 10B). These results indicated that a 13 amino acidslong peptide sequence ranging from K-157 to M-169 (KRLDVCRAKMGYM) of theUL128 protein is necessary and sufficient for efficient 13B5 binding. Toinvestigate further whether additional amino acid residues localized atthe N-terminus of K-157 of UL128 are critical for 13B5 recognition, 13B5binding to peptides composed of the defined minimal 13B5 binding sitewas compared with peptides comprising the 13B5 binding site and one ortwo additional amino acids (K-155 and H-156) of UL128 added to theN-terminus. As shown in FIG. 10C, addition of the N-terminal amino acids(KH) to the 13B5 target sequence only minimally improved binding of 13B5antibody, suggesting that these amino acids are not critical for 13B5binding but they may slightly improve the interaction of the 13B5antibody and its target site. In sum, these results suggest that NAb13B5 targets a 13 amino acid long linear epitope sequence at theC-terminus of UL128 that is composed of residues 157-KRLDVCRAKMGYM-169of the protein.

Most Residues of the 1385 Target Site within UL128 are Critical forAntibody Binding.

In order to define the amino acid residues of the 13B5 target sitewithin UL128 that are critical for antibody binding, each residue of thedefined 13 amino acid long 13B5 binding site was serially substitutedwith alanine residues, and the influence of these changes on 13B5binding was evaluated. FIG. 10D shows the individual sequences of themutated peptides as well as the testing of 13B5 binding to thesepeptides via ELISA. Note that peptide K13M is identical to the originalsequence of the 13B5 binding site within UL128 (157-KRLDVCRAKMGYM-169).This peptide comprises an internal alanine and hence was used as controlin the ELISA for testing 13B5 binding. Consistent with results obtainedwith truncation libraries, alanine substitution of the N-terminal K andC-terminal Y or M of K13M significantly reduced binding of the 13B5antibody. Similarly, substituting most of the internal amino acids (C,R, K, M, and G) of K13M with alanine residues resulted in dramaticallyreduced or complete loss of 13B5 binding. In contrast, 13B5 binding wasnot impaired when one of the internal residues L, D, and V of K13M weresubstituted. In addition, substitution of L or D within K13M slightlyincreased 13B5 binding when compared to the original K13M sequence,suggesting that these amino acid substitutions slightly improved theinteraction of 13B5 and its target sequence. Interestingly, the internalC within K13M that appeared essential for 13B5 binding corresponds toamino acid C-162 of UL128 that has previously been shown to form adisulfide bridge with gL in the PC (4). This suggests that NAb 13B5targets a sequence within UL128 that is critical for interaction of thePC subunits. In sum, these results indicate that most of the amino acidsof the 13B5 target site within UL128 are directly involved in theinteraction with the 13B5 antibody, while three residues within thetarget site appear not to be involved directly in 13B5 binding.

Example 12. Validation of Small Peptides

Peptide Construction Based on the 1385 Binding Site to Test AntibodyInduction.

To determine whether the UL128 binding site of NAb 13B5 is aneutralizing determinant, keyhole limpet hemocyanin (KLH)-coupledpeptides based on the 13B5 binding site were evaluated forimmunogenicity to elicit NAb in mice. For this, three differentKLH-coupled peptide constructs were generated based on the 13B5 targetsequence. In one construct, termed KLH-K15M (i.e., KLH-coupled SEQ IDNO: 179), KLH was coupled to the minimal 13B5 target sequence (K13M) viathe existing internal C-162, and two additional residues of UL128 wereadded to the peptide C-terminus, which appeared to increase slightly thebinding of 13B5 antibody. The second construct named KLH-K14CS (i.e.,KLH-coupled SEQ ID NO: 180) was generated by coupling of KLH via aC-terminally added C residue to K13M in which the internal C-162 of theminimal 13B5 target sequence was substituted with a serine. The thirdconstruct, termed KLH-K16CS (i.e., KLH-coupled SEQ ID NO: 181), wasgenerated in a similar way as the second construct (KLH-K14CS), exceptthat it comprised two additional amino acid residues of UL128 at theN-terminus similar to the first construct (KLH-K15M). C to S amino acidsubstitutions in the second and third construct (KLH-K14CS, KLH-K16CS)were chosen because of the similarity in steric occupancy between thesetwo residues. As shown in FIG. 11A, all KLH construct showed strongbinding of 13B5 antibody, indicating that the KLH coupling and aminosubstitutions did not alter the interaction of the 13B5 target sequencewith the 13B5 antibody. In addition, serine substitution of the internalC-162 in peptide K13M with minimal 13B5 target site—not coupled toKLH—and subsequent addition of a C-terminal cysteine did not result in adecrease of 13B5 binding when compared with the original K13M peptide.However, C-1B2 to serine substitution in K13M without adding aC-terminal cysteine dramatically decreased 13B5 binding, which isconsistent with our findings with the alanine scanning procedure (seeFIG. 10D). This suggests that the cysteine residue of the 13B5 targetsite is essential for 13B5 binding, but its location within the targetsite at position 1B2 of the UL128 protein sequence might not beabsolutely fundamental for the antibody binding. As control, a peptideconstruct C-terminally coupled to KLH (KLH-38) consisting of librarypeptide 38 that was only partially overlapping with the 13B5 target sitewas generated. This peptide was similar to a recently tested UL128peptide that has failed to stimulate NAb in rabbits (5). KLH-38 wasunable to bind 13B5 antibody, supporting its use as a control construct.In sum, these results indicated that all KLH-coupled peptide constructspresented intact 13B5 target sites.

Peptides Based on the 1385 Target Site have Ability to Elicit NAb inMice.

For testing whether the generated KLH coupled peptide constructs basedon the 13B5 target site (KLH-K15M, KLH-K14CS, KLH-K16CS) have ability toelicit NAb in vivo, Balb/c mice were intraperitoneally immunized threetimes four weeks apart with the peptide constructs admixed in Freund'sadjuvant. Serum binding antibodies and NAb were determined one weekbefore and three weeks after each immunization. Binding antibodies ofthe individual groups were determined via ELISA using peptides as targetantigens that were used for immunization (FIG. 11B). NAb titer at which50% infection (NT50) was inhibited were determined by microneutralization assay using ARPE-19 EC as cell substrate and HCMV strainTB40/E for infection (FIG. 11C). For control, mice were immunized withKLH only or KLH-38 with C-terminal UL128 peptide sequence that onlypartially overlapped with the 13B5 target site. As shown in FIG. 11B,all mice immunized with the peptide constructs developed bindingantibodies against the peptide sequence with which they were vaccinatedafter the first immunization. These responses were not or only minimallyboosted by a second and third immunization. In addition, Western Blotanalysis of UL128 expressed from Ad vectors showed that mice immunizedwith KLH-K15M, KLH-K14CS, KLH-K16CS developed antibodies that recognizeddenatured UL128. NAb responses with NT50 titers ranging from 200 to 300were detectable after the first immunization in only two out of fiveanimals in each vaccine group immunized with KLH-K14CS or KLH-K16CS(FIG. 11C). These responses were boosted in only one animal in each ofthe KLH-K14CS or KLH-K16CS vaccine groups, reaching NT50 titers of 800to 1200. These titers remained stable after the third immunization. Inthe two other animals that developed NAb after the first immunization,NAb declined and were undetectable after the booster immunizations. Noneof the animals immunized with the peptide construct KLH-K15M, controlconstruct KLH-38, or KLH only developed NAb. These results indicate thatpeptides based on the 13B5 target site in which the internal cysteineresidue was substituted by a serine and KLH was coupled via anadditional cysteine residue to the C-terminus had ability to elicit NAbin mice. In contrast, peptides composed of the 13B5 target site thatwere coupled to KLH via the existing internal C-1B2 did not showimmunogenicity for NAb induction. These results support that theidentified 13B5 target site constitutes a neutralizing epitope withinUL128.

REFERENCES

The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entireties, as if fully set forthherein.

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What is claimed is:
 1. A vaccine-derived neutralizing antibody (NAb)against cytomegalovirus (CMV) comprising: a variable heavy regioncomprising a CDR1_(VH) sequence, a CDR2_(VH) sequence, and a CDR3_(VH)sequence; and a variable light region comprising a CDR1_(VL) sequence, aCDR2_(VL) sequence, and a CDR3_(VL) sequence; wherein thevaccine-derived NAb is produced in response to a recombinant CMVpentameric complex comprising gH, gL, UL128, UL130, and UL131A subunits.2. The vaccine-derived NAb of claim 1, wherein the vaccine-derived NAbis similar or identical to a NAb induced in a subject naturally infectedwith CMV in one or more properties selected from the group consisting ofcell-type specificity, neutralization potency, minimal antigenrecognition, and frequency to recognize antigenic sites.
 3. Thevaccine-derived NAb of claim 1, wherein the vaccine-derived NAb preventscell-to-cell spread of CMV, syncytia formation in epithelial cells, orboth.
 4. The vaccine-derived NAb of claim 1, wherein the vaccine-derivedNAb has a positive correlation between neutralizing potency and bindingaffinity of one or more cell surface subunits of the pentameric complex.5. The vaccine-derived NAb of claim 1, wherein the vaccine-derived NAbspecifically binds one or more linear epitopes on the recombinant CMVpentameric complex.
 6. The vaccine-derived NAb of claim 5, wherein thelinear epitope is on UL128 of the recombinant CMV pentameric complex. 7.The vaccine-derived NAb of claim 6, wherein the linear epitope on UL128comprises an amino acid sequence represented by SEQ ID NO: 177(KRLDVCRAKMGYM).
 8. The vaccine-derived NAb of claim 5, wherein thelinear epitope is on gH of the recombinant CMV pentameric complex. 9.The vaccine-derived NAb of claim 8, wherein the vaccine-derived NAbneutralizes CMV infection of epithelial cells but not CMV infection offibroblasts.
 10. The vaccine-derived NAb of claim 1, wherein thevaccine-derived NAb specifically binds one or more conformationalepitopes on the recombinant CMV pentameric complex.
 11. Thevaccine-derived NAb of claim 1, wherein the vaccine-derived NAbspecifically binds to one or more conformational epitopes composed ofUL128/UL130/UL131A or UL130/UL131A subunits of the recombinant CMVpentameric complex.
 12. The vaccine-derived NAb of claim 11, wherein thevaccine-derived NAb neutralizes CMV infection of epithelial cells,endothelial cells, primary placental cytotrophoblast cells or acombination thereof.
 13. The vaccine-derived NAb of claim 1, wherein thevaccine-derived NAb specifically binds to one or more conformationalepitopes on gH or gH/gL.
 14. The vaccine-derived NAb of claim 13,wherein the vaccine-derived NAb prevents CMV infection of fibroblasts,epithelial cells, endothelial cells, cytotrophoblasts or a combinationthereof.
 15. The vaccine-derived NAb of claim 1, wherein the CDR1_(VH)sequence is selected from the group consisting of SEQ ID NOs: 3, 11, 19,27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123, 131, 139, 147,155, 163, 171, and sequences sharing at least 90% identity to SEQ IDNOs: 3, 11, 19, 27, 35, 43, 51, 59, 67, 75, 83, 91, 99, 107, 115, 123,131, 139, 147, 155, 163,
 171. 16. The vaccine-derived NAb of claim 1,wherein the CDR2_(VH) sequence is selected from the group consisting ofSEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84, 92, 100, 108,116, 124, 132, 140, 148, 156, 164, 172, and sequences sharing at least90% identity to SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 76, 84,92, 100, 108, 116, 124, 132, 140, 148, 156, 164,
 172. 17. Thevaccine-derived NAb of claim 1, wherein the CDR3_(VH) sequence isselected from the group consisting of SEQ ID NOs: 5, 13, 21, 29, 37, 45,53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165,173, and sequences sharing at least 90% identity to SEQ ID NOs: 5, 13,21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141,149, 157, 165,
 173. 18. The vaccine-derived NAb of claim 1, wherein theCDR1_(VL) sequence is selected from the group consisting of SEQ ID NOs:6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110, 118, 126, 134,142, 150, 158, 166, 174 and sequences sharing at least 90% identity toSEQ ID NOs: 6, 14, 22, 30, 38, 46, 54, 62, 70, 78, 86, 94, 102, 110,118, 126, 134, 142, 150, 158, 166,
 174. 19. The vaccine-derived NAb ofclaim 1, wherein the CDR2_(VL) sequence is selected from the groupconsisting of SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95,103, 111, 119, 127, 135, 143, 151, 159, 167, 175 and sequences sharingat least 90% identity to SEQ ID NOs: 7, 15, 23, 31, 39, 47, 55, 63, 71,79, 87, 95, 103, 111, 119, 127, 135, 143, 151, 159, 167,
 175. 20. Thevaccine-derived NAb of claim 1, wherein the CDR3_(VL) sequence isselected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48,56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152, 160, 168, 176and sequences sharing at least 90% identity to SEQ ID NOs: 8, 16, 24,32, 40, 48, 56, 64, 72, 80, 88, 96, 104, 112, 120, 128, 136, 144, 152,160, 168,
 176. 21. A composition for treating or preventing CMVinfection comprising a vaccine-derived neutralizing antibody (NAb)against cytomegalovirus (CMV), which vaccine-derived NAb comprises: avariable heavy region comprising a CDR1_(VH) sequence, a CDR2_(VH)sequence, and a CDR3_(VH) sequence; and a variable light regioncomprising a CDR1_(VL) sequence, a CDR2_(VL) sequence, and a CDR3_(VL)sequence; wherein the vaccine-derived NAb is produced in response to arecombinant CMV pentameric complex comprising gH, gL, UL128, UL130, andUL131A subunits.
 22. The composition of claim 21, wherein thevaccine-derived NAb is humanized.
 23. A peptide comprising a linearepitope on UL128, wherein the peptide comprises at least one cysteineresidue and specifically binds to a neutralizing antibody of CMV. 24.The peptide of claim 23, wherein the peptide has a size of 10 aminoacids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids,15 amino acids, 16 amino acids, 17 amino acids, or 18 amino acids. 25.The peptide of claim 23, wherein the peptide has an amino acid sequenceselected from the group consisting of KRLDVCRAKMGYM (SEQ ID NO: 177),HKRLDVCRAKMGYM (SEQ ID NO: 178), KHKRLDVCRAKMGYM (SEQ ID NO: 179),KRLDVSRAKMGYMC (SEQ ID NO: 180), KHKRLDVSRAKMGYMC (SEQ ID NO: 181), anda sequence which is at least 80%, at least 85%, at least 90%, or atleast 95% identical to SEQ ID NOs. 177-181.
 26. A vaccine compositionfor preventing CMV infection comprising one or more peptides of claims23-25.
 27. A method of producing a vaccine-derived neutralizing antibody(NAb) against cytomegalovirus (CMV) comprising: administering to asubject an effective amount of a recombinant CMV pentameric complexcomprising gH, gL, UL128, UL130, and UL131A subunits; deriving ahybridoma from the subject; and isolating the NAb from the hybridoma.28. A method of detecting the presence of a CMV antigen in a biologicalsample or a cell culture comprising contacting the sample or the cellculture with a vaccine-derived neutralizing antibody (NAb) againstcytomegalovirus (CMV), which vaccine-derived NAb comprises: a variableheavy region comprising a CDR1_(VH) sequence, a CDR2_(VH) sequence, anda CDR3_(VH) sequence; and a variable light region comprising a CDR1_(VL)sequence, a CDR2_(VL) sequence, and a CDR3_(VL) sequence; wherein thevaccine-derived NAb is produced in response to a recombinant CMVpentameric complex comprising gH, gL, UL128, UL130, and UL131A subunits.29. A method of treating or preventing CMV infection in a subject,comprising administering to the subject an effective amount of acomposition comprising a vaccine-derived neutralizing antibody (NAb)against cytomegalovirus (CMV), which vaccine-derived NAb comprises: avariable heavy region comprising a CDR1_(VH) sequence, a CDR2_(VH)sequence, and a CDR3_(VH) sequence; and a variable light regioncomprising a CDR1_(VL) sequence, a CDR2_(VL) sequence, and a CDR3_(VL)sequence; wherein the vaccine-derived NAb is produced in response to arecombinant CMV pentameric complex comprising gH, gL, UL128, UL130, andUL131A subunits.